Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins

ABSTRACT

The present invention pertains to a method for delivering a neuropharmaceutical agent across the blood brain barrier to the brain of a host. The method comprises administering to the host a therapeutically effective amount of a ligand-neuropharmaceutical agent fusion protein wherein the ligand is reactive with a brain capillary endothelial cell receptor. Other aspects of this invention include a delivery system comprising a ligand reactive with a brain capillary endothelial cell receptor which has formed a fusion protein with a neuropharmaceutical agent. The fusion proteins are also aspects of this invention.

RELATED APPLICATIONS

This Application is the U.S. National Phase of PCT/US94/08000, filedJul. 18, 1994, which is a Continuation-in-Part of U.S. Ser. No.08/094,534, filed Jul. 16, 1993, now U.S. Pat. No. 5,672,683 which isContinuation-in-Part of Ser. No. 07/999,803, filed Nov. 20, 1992 (nowabandoned), which is a Divisional of Ser. No. 07/846,830, filed Mar. 6,1992 (now U.S. Pat. No. 5,182,107), which is a Continuation-in-Part ofPCT/US90/05077, filed Sep. 7, 1990, which is a Continuation-in-Part ofU.S. Ser. No. 07/404,089, filed Sep. 7, 1989 (now U.S. Pat. No.5,154,924, issued Oct. 13, 1992).

BACKGROUND

The capillaries that supply blood to the tissues of the brain constitutethe blood brain barrier (Goldstein et al. (1986) Scientific American255:74-83; Pardridge, W. M. (1986) Endocrin. Rev. 7:314-330). Theendothelial cells which form the brain capillaries are different fromthose found in other tissues in the body. Brain capillary endothelialcells are joined together by tight inter-cellular junctions which form acontinuous wall against the passive movement of substances from theblood to the brain. These cells are also different in that they have fewpinocytic vesicles which in other tissues allow somewhat unselectivetransport across the capillary wall. Also lacking are continuous gaps orchannels running through the cells which would allow unrestrictedpassage.

The blood-brain barrier functions to ensure that the environment of thebrain is constantly controlled. The levels of various substances in theblood, such as hormones, amino acids and ions, undergo frequent smallfluctuations which can be brought about by activities such as eating andexercise (Goldstein et al., cited supra). If the brain were notprotected by the blood brain barrier from these variations in serumcomposition, the result could be uncontrolled neural activity.

The isolation of the brain from the bloodstream is not complete. If thiswere the case, the brain would be unable to function properly due to alack of nutrients and because of the need to exchange chemicals with therest of the body. The presence of specific transport systems within thecapillary endothelial cells assures that the brain receives, in acontrolled manner, all of the compounds required for normal growth andfunction. In many instances, these transport systems consist ofmembrane-associated receptors which, upon binding of their respectiveligand, are internalized by the cell (Pardridge, W. M., cited supra).Vesicles containing the receptor-ligand complex then migrate to theabluminal surface of the endothelial cell where the ligand is released.

The problem posed by the blood-brain barrier is that, in the process ofprotecting the brain, it excludes many potentially useful therapeuticagents. Presently, only substances which are sufficiently lipophilic canpenetrate the blood-brain barrier (Goldstein et al., cited supra;Pardridge, W. M., cited supra). Some drugs can be modified to make themmore lipophilic and thereby increase their ability to cross the bloodbrain barrier. However, each modification has to be tested individuallyon each drug and the modification can alter the activity of the drug.The modification can also have a very general effect in that it willincrease the ability of the compound to cross all cellular membranes,not only those of brain capillary endothelial cells.

SUMMARY OF THE INVENTION

The present invention pertains to a method for delivering aneuropharmaceutical agent across the blood brain barrier to the brain ofa host. The method comprises administering to the host aligand-neuropharmaceutical agent fusion protein wherein the ligand isreactive with a brain capillary endothelial cell receptor. The ligand ofthe fusion protein is an intact ligand to a brain capillary endothelialcell receptor or a receptor-binding fragment thereof. Alternatively, theligand can be an antibody or immunoreactive fragment thereof that isreactive with a brain capillary endothelial cell receptor. Theneuropharmaceutical agent of the fusion protein is a protein,polypeptide or peptide. The fusion protein is administered underconditions whereby binding of the ligand to a receptor on a braincapillary endothelial cell occurs and the neuropharmaceutical agent istransferred across the blood brain barrier in a pharmaceutically activeform and in a therapeutically effective amount.

The present invention also pertains to a delivery system comprising aligand-neuropharmaceutical agent fusion protein wherein the ligand isreactive with a brain capillary endothelial cell receptor. This deliverysystem transports the neuropharmaceutical agent across the blood brainbarrier in a pharmaceutically active form when the fusion protein isadministered in vivo. The present invention also pertains to the fusionproteins themselves which have both ligand binding andneuropharmaceutical characteristics.

Fusion proteins which include a brain capillary endothelial cellreceptor ligand and an antibody, or immunoreactive fragment thereof,that is itself reactive with a brain capillary endothelial cell receptorare other aspects of the present invention. In these aspects,neuropharmaceutical agents can be conjugated to the fusion proteins bycleavable or noncleavable linkers for transport of these agents acrossthe blood brain barrier. Also pertaining to the present invention are adelivery system incorporating these fusion proteins and a method fordelivering the neuropharmaceutical agent across the blood brain barrierin a pharmaceutically active form by administering to the host a fusionprotein-neuropharmaceutical agent conjugate in a therapeuticallyeffective amount.

Presently available means for delivering neuropharmaceutical agents tothe brain are limited in that they are invasive. The delivery system ofthe present invention is non-invasive and can utilize readily availableligands reactive with brain capillary endothelial cell receptors ascarriers for neuropharmaceutical agents. The delivery system isadvantageous in that the ligands, when formed as part of a fusionprotein with neuropharmaceutical agents, are capable of transporting theneuropharmaceutical agents across the blood brain barrier without beingsusceptible to premature release of the neuropharmaceutical agent priorto reaching the brain side of the blood brain barrier. The deliverysystem is similarly advantageous when the neuropharmaceutical agent isconjugated to the fusion protein by a noncleavable bond.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of rat brain uptake of ¹⁴ C-labelledmurine monoclonal antibody (0X-26) to rat transferrin receptor in ratswhere the percent injected dose of radiolabelled antibody per brain andper 55 μl of blood is plotted versus time post-injection.

FIG. 2 is a histogram illustrating time dependent changes in thedisposition of radiolabelled OX-26 between brain parenchyma andvasculature.

FIG. 3 is a histogram illustrating the biodistribution of antibody 128.1and control IgG in a cynomolgus monkey.

FIG. 4 is a restriction enzyme map of bacterial plasmid pAT3442 whichadditionally shows the human IgG3 and transferrin gene regions.

FIG. 5A is a restriction enzyme map of the CH1-hinge-transferrin regionof clone pATX.

FIG. 5B is a restriction enzyme map of the CH1-hinge-transferrin regionof clone pATXX.

FIG. 5C is a restriction enzyme map of the pre-pro NGF-hinge-transferrinregion of clone pATXXNGF.

FIG. 6 is a restriction enzyme map of clones pUCNGF1 and pUCNGF2 whichcontain the NGF gene in opposite orientations.

FIG. 7 is a set of restriction enzyme maps that depict the formation ofplasmid pcDNAI/AmpNHT with fragments from clones pUCNGF2 and pATXXNGF.

FIG. 8 is a restriction enzyme map of clone CD5lneg1.

FIG. 9A-9B is the DNA sequence between the XhoI and EagI sites of cloneCD5lneg1, where the coding sequences for the CD5 Leader and IgG1 Exons1, 2 and 3 are displayed in larger, bold characters (Seq.I.D.NO.8).

FIG. 10 is a graphic representation of the competition between eitherrecombinant human transferrin or NHT fusion protein and radioactivelylabeled transferrin when binding to human placental transferrinreceptors. FIG. 11A is a graphic representation of the inducing effectof NGF on the sprouting of neurites from PC12 cells.

FIG. 11B is a graphic representation of the inducing effect of NHTfusion protein on the sprouting of neurites from PC12 cells.

FIG. 12A is a restriction enzyme map of plasmids D1 and d1.

FIG. 12B is a restriction enzyme map of plasmids C4 and C*.

FIG. 12C is a restriction enzyme map of plasmid C4-NHT.

FIG. 12D is a restriction enzyme map of plasmid g*.

FIG. 12E is a restriction enzyme map of plasmid H45.

FIG. 12F is a restriction enzyme map of plasmid gH.

DETAILED DESCRIPTION

The method for delivering a neuropharmaceutical agent across the bloodbrain barrier to the brain of a host comprises administering to the hosta ligand-neuropharmaceutical agent fusion protein wherein the ligand isreactive with a receptor present on a brain capillary endothelial cell.The method is conducted under conditions whereby the ligand binds to thereceptor on the brain capillary endothelial cell and theneuropharmaceutical agent is transferred across the blood brain barrierin a pharmaceutically active form and in a therapeutically effectiveamount.

The ligand-neuropharmaceutical agent fusion protein, which has bothligand binding and neuropharmaceutical characteristics, can be producedas a contiguous protein by using genetic engineering techniques. Geneconstructs can be prepared comprising DNA encoding the ligand fused toDNA encoding the protein, polypeptide or peptide to be delivered acrossthe blood brain barrier. The ligand coding sequence and the agent codingsequence are inserted in the expression vectors in a suitable manner forproper expression of the desired fusion protein. The gene fusion isexpressed as a contiguous protein molecule containing both a ligandportion and a neuropharmaceutical agent portion. For example, sequencesencoding neurotrophic agents such as NGF (nerve growth factor) or CNTF(ciliary neurotrophic factor) can be fused with the sequence encodingtransferrin to create chimeric polypeptides that will be expressed andsubsequently transported across the BBB via the transferrin receptor.

The genetic engineering techniques are often used to insert linker DNAsequences between the ligand and the neuropharmaceutical agent DNAencoding sequences. These linker DNA sequences can be expressed as partof the fusion protein. For example, specific segments of the constantregion of an antibody, including the hinge region, can be insertedbetween the ligand and the neuropharmaceutical agent. These expressedinsertions serve to separate the ligand from the neuropharmaceuticalagent and may facilitate the proper folding of the expressed ligand oragent into its proper conformation. When the insertions are segmentsfrom the constant region of antibodies that are syngeneic to the host,they have the added advantage of having reduced immunogenicity whenadministered.

The host can be an animal susceptible to a neurological disorder (i.e.,an animal having a brain). Examples of hosts include mammals such ashumans, domestic animals (e.g., dog, cat, cow or horse), mice and rats.

The neuropharmaceutical agent can be an agent having a therapeutic orprophylactic effect on a neurological disorder or any condition whichaffects biological functioning of the central nervous system. Examplesof neurological disorders include cancer (e.g. brain tumors), AcquiredImmune Deficiency Syndrome (AIDS), stroke, epilepsy, Parkinson'sdisease, autoimmune diseases such as multiple sclerosis,neurodegenerative disease, trauma, depression, Alzheimer's disease,migraine, pain, or a seizure disorder. Classes of neuropharmaceuticalagents which can be used in this invention include proteins andpolypeptides used to treat or prevent a neurological disorder. Examplesof proteins include growth factors (e.g. nerve growth factor (NGF)),ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor(BDNF), glial cell-line derived neurotrophic factor (GDNF),neurotrophins 3,4 and 5 (NT-3,4 and 5) or fibroblast growth factor(FGF), lymphokines or cytokines (e.g. interferon or interleukins (IL-2))or antagonists thereof, CD4 and superoxide dismutase (including solubleportions thereof), dopamine decarboxylase and tricosanthin. Examples ofpolypeptides include somatostatin analogues and enkephalinaseinhibitors.

The ligand of the fusion protein is any polypeptide or protein that iscapable of binding with specificity to a receptor on brain capillaryendothelial cells. These receptors are normally located on the luminalsurfaces of these endothelial-cells when they line the inner portion ofthe brain blood vessels. A particularly preferred ligand family istransferrin and any transferrin derivatives which retain transferrinreceptor-binding activity.

Serum transferrin is a monomeric glycoprotein with a molecular weight of80,000 daltons that binds iron in the circulation and transports it tothe various tissues(Aisen et al. (1980) Ann. Rev. Biochem. 49: 357-393;MacGillivray et al. (1981) J. Biol. Chem. 258: 3543-3553). The uptake ofiron by individual cells is mediated by the transferrin receptor, anintegral membrane glycoprotein consisting of two identical 95,000 daltonsubunits that are linked by a disulfide bond. The number of receptors onthe surface of a cell appears to correlate with cellular proliferation,with the highest number being on actively growing cells and the lowestbeing on resting and terminally differentiated cells. Jeffries et al.(Nature 312, pp. 167-168 (November 1984)) used monoclonal antibodies toshow that brain capillary endothelial cells have a high density oftransferrin receptors on their cell surface.

Fusion proteins comprising ligands and neuropharmaceutical agents canalso be prepared where the ligands are reactive with other receptors,besides the transferrin receptor, which can also mediate the endocytoticor transcytotic process of transporting macromolecules across theblood-brain barrier. These receptors are also on the cell surface of theendothelial cells which line brain vessels.

Among the receptor types are those that react with insulin-like growthfactors 1 or 2 (IGF 1 or 2) or insulin and derivatives of these ligandswhich retain receptor-binding activity. The therapeutic agents which canbe conjugated to the ligands include the above-mentioned proteins suchas nerve growth factor, ciliary neurotrophic factor, brain-derivedneurotrophic factor, superoxide dismutase, CD-4 or anti-amyloidantibody.

The term receptor is intended to encompass the entire receptor orligand-binding portions thereof. These portions of the receptorparticularly include those regions sufficient for specific binding ofthe ligand to occur.

Ligands which can bind with specificity to brain capillary endothelialcell receptors include antibodies or antibody fragments that can bindwith these receptors. These antibodies or antibody fragments are ascapable of binding to the brain capillary endothelial cell receptors asthe nominal receptor ligands. Upon binding of the antibodies to thereceptors, transferal of the antibody and any attached agent across theblood brain barrier occurs. The agent can be attached by any acceptablemeans for joining the antibody and agent such that the agent can betransferred across the blood brain barrier in a pharmaceutically activeform. In preferred embodiments, the attached substance is aneuropharmaceutical agent and the antibody or antibody fragment forms afusion protein with the agent. The antibody has replaced the nominalligand, such as transferrin or receptor-binding derivatives oftransferrin, in these embodiments.

In other embodiments, an antibody or antibody fragment, which isimmunoreactive with a brain capillary endothelial cell receptor, and asecond ligand, which is reactive with the same or a different receptortype on the brain capillary endothelial cells, are joined together toform a fusion protein. The second ligand can be a second antibody or,more preferably, a nominal ligand such as transferrin, IGFI, IGF2 orinsulin. Conversely, the two ligands of the fusion protein can be twonominal ligands. These fusion proteins have the advantage of possessingthe capacity of interacting twice as readily with brain capillaryendothelial cell receptors than the fusion proteins of the presentinvention which have only one ligand. These fusion proteins can belinked by either genetic or chemical conjugation means toneuropharmaceutical agents for transferal of these agents across theblood brain barrier in a pharmaceutically active form.

When fusion proteins comprising an antibody and a second ligand areused, the range of neuropharmaceutical agents that can be transferredacross the blood brain barrier is markedly increased. In addition toproteins and polypeptides, other substances that can be linked to thefusion proteins include antibiotics, adrenergic agents, anticonvulsants,nucleotide analogs, chemotherapeutic agents, anti-inflammatory agentsand anti-trauma agents used to treat or prevent a disease of the brainor central nervous system (CNS). Examples of antibiotics includeamphotericin B, gentamicin sulfate and pyrimethamine. Examples ofadrenergic agents (including blockers) include dopamine and atenolol.Examples of chemotherapeutic agents include adriamycin, methotrexate,cyclophosphamide, etoposide and carboplatin. An example of ananticonvulsant which can be used is valproate and an anti-trauma agentwhich can be used is superoxide dismutase. Nucleotide analogs which canbe used include azidothymidine (hereinafter AZT), dideoxyinosine (ddI)and dideoxycytodine (ddC). Examples of anti-inflammatory agents includetumor necrosis factor (TNF) and transforming growth factor (TGFβ).

The neuropharmaceutical agent can be linked to the antibody--secondligand fusion protein using chemical conjugation techniques. Generally,the link is made via an amine or a sulfhydryl group. The link can be acleavable link or non-cleavable link depending upon whether theneuropharmaceutical agent is more effective when released in its nativeform or whether the pharmaceutical activity of the agent can bemaintained while linked to the fusion protein. The determination ofwhether to use a cleavable or non-cleavable linker can be made withoutundue experimentation by measuring the activity of the drug in bothnative and linked forms or for some drugs can be determined based onknown activities of the drug in both the native and linked form.

For some cases involving the delivery of protein or peptide agents tothe brain, release of the free protein or peptide may not be necessaryif the biologically active portion of the protein or peptide agent isunaffected by its attachment to the fusion protein. As a result,antibody-protein or antibody-peptide conjugates can be constructed usingnoncleavable linkers.

Examples of non-cleavable linker systems which can be used in theseembodiments include the carbodiimide (EDC), the sulfhydryl-maleimide,and the periodate systems. In the carbodiimide system, a water solublecarbodiimide reacts with carboxylic acid groups on proteins andactivates the carboxyl group. The carboxyl group is coupled to an aminogroup of the second protein. The result of this reaction is anoncleavable amide bond between two proteins.

In the sulfhydryl-maleimide system, a sulfhydryl group is introducedonto an amine group of one of the proteins using a compound such asTraut's reagent. The other protein is reacted with an NHS ester (such asgamma-maleimidobutyric acid NHS ester (GMBS)) to form a maleimidederivative that is reactive with sulfhydryl groups. The two modifiedproteins are then reacted to form a covalent linkage that isnoncleavable.

Periodate coupling requires the presence of oligosaccharide groups oneither the fusion protein carrier or the protein to be delivered. Ifthese groups are available on the protein to be delivered (as in thecase of horseradish peroxidase (HRP)), an active aldehyde is formed onthe protein to be delivered which can react with an amino group on thecarrier. It is also possible to form active aldehyde groups from thecarbohydrate groups present on antibody molecules. These groups can thenbe reacted with amino groups on the protein to be delivered generating astable conjugate. Alternatively, the periodate oxidized antibody can bereacted with a hydrazide derivative of a protein to be delivered whichwill also yield a stable conjugate.

Cleavable linkers can be used to link neuropharmaceutical agents whichare to be deposited in the brain or when a non-cleavable linker altersthe activity of a neuropharmaceutical agent. Examples of cleavablelinkers include the acid labile linkers described in copending patentapplication Ser. No. 07/308,960 filed Feb. 6, 1989, now U.S. Pat. No.5,144,011 the contents of which are hereby incorporated by reference.Acid labile linkers include disulfides such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP; Pharmacia),cis-aconitic acid, cis-carboxylic alkadienes, cis-carboxylicalkatrienes, and poly-maleic anhydrides. Other cleavable linkers arelinkers capable of attaching to primary alcohol groups. Examples ofneuropharmaceutical agents which can be linked via a cleavable linkinclude AZT, ddI, ddC, adriamycin, amphotericin B, pyrimethamine,valproate, methotrexate, cyclophosphamide, carboplatin and superoxidedismutase. The noncleavable linkers used generally to link proteins tothe antibody can also be used to link other neuropharmaceutical agentsto the antibody.

SPDP is a heterobifunctional crosslinking reagent that introducesthiol-reactive groups into either the monoclonal antibody or theneuropharmaceutical agent. The thiol-reactive group reacts with a freesulfhydryl group forming a disulfide bond.

In addition to covalent bonding, conjugates can be formed employingnon-covalent bonds, such as those formed with bifunctional antibodies,ionic bonds, hydrogen bonds, hydrophobic interactions, etc. Theimportant consideration is that the conjugate bond be strong enough toresult in passage of the conjugate through the blood-brain barrier.

Antibodies which can be used within this invention are reactive with areceptor on a brain capillary endothelial cell. The term antibody isintended to encompass both polyclonal and monoclonal antibodies. Thepreferred antibody is a monoclonal antibody reactive with a braincapillary endothelial cell receptor such as a transferrin receptor. Theterm antibody is also intended to encompass mixtures of more than oneantibody reactive with a transferrin receptor (e.g., a cocktail ofdifferent types of monoclonal antibodies reactive with a transferrinreceptor), each of which is joined to a neuropharmaceutical agent oranother ligand to form a fusion protein. The term antibody is furtherintended to encompass whole antibodies, biologically functionalfragments thereof, and chimeric antibodies comprising portions from morethan one species, bifunctional antibodies, etc. Biologically functionalantibody fragments which can be used are those fragments sufficient forbinding of the antibody fragment to the brain capillary endothelial cellreceptor to occur.

The chimeric antibodies can comprise portions derived from two differentspecies (e.g., human constant region and murine variable or bindingregion). The portions derived from two different species can be joinedtogether chemically by conventional techniques or can be prepared asfusion proteins using genetic engineering techniques. In addition, DNAencoding the proteins of both the light chain and heavy chain portionsof the chimeric antibody can be expressed together as fusion proteins.

Such chimeric antibodies can readily be adapted to being part of thefusion proteins of this invention. The DNA which contains the variableregion coding sequence can be fused to DNA which contains theneuropharmaceutical agent coding sequence for subsequent expression as afusion protein. Likewise, the DNA which contains the variable regioncoding sequence can be fused to DNA which contains the coding sequenceof a second ligand, if such an expressed fusion protein is desired. Thechimeric antibodies comprising constant and variable region portionsfrom two different species can easily be converted to fusion proteins ofthis invention by inserting DNA encoding a neuropharmaceutical agent orDNA encoding another ligand after a specific portion of constant regionencoding DNA. The subsequently expressed fusion protein will thencontain the variable region from one species, a desired portion of theconstant region from another species and a second ligand or the agent tobe transferred across the blood brain barrier.

Monoclonal antibodies reactive with at least a portion of thetransferrin receptor can be obtained (e.g., 0X-26, B3/25 (Omary et al.(1980) Nature 286: 888-891), T56/14 (Gatter et al. (1983) J. Clin. Path.36: 539-545; Jefferies et al. Immunology (1985) 54: 333-341), OKT-9(Sutherland et al. (1981) Proc. Natl. Acad. Sci. USA 78: 4515-4519),L5.1 (Rovera, C. (1982) Blood 59: 671-678), 5E-9 (Haynes et al.(1981) J.Immunol. 127: 347-351), RI7 217 (Trowbridge et al. Proc. Natl. Acad.Sci. USA 78: 3039 (1981) and T58/30 (Omary et al. cited supra) or can beproduced using somatic cell hybridization techniques (Kohler andMilstein (1975) Nature 256: 495-497) or by other techniques. In atypical hybridization procedure, a crude or purified protein or peptidecomprising at least a portion of the transferrin receptor can be used asthe immunogen. An animal is vaccinated with the immunogen to obtainanti-transferrin receptor antibody-producing spleen cells. The speciesof animal immunized will vary depending on the species of monoclonalantibody desired. An antibody-producing cell is fused with animmortalizing cell (e.g. myeloma cell) to create a hybridoma capable ofsecreting anti-transferrin receptor antibodies. The unfused residualantibody-producing cells and immortalizing cells are eliminated.Hybridomas producing the anti-transferrin receptor antibodies areselected using conventional techniques and the selected anti-transferrinreceptor antibody producing hybridomas are cloned and cultured. Similarsomatic cell hybridization techniques can be used to produce hybridomasthat secrete monoclonal antibodies immunoreactive with other braincapillary endothelial cell receptors.

Polyclonal antibodies can be prepared by immunizing an animal with acrude or purified protein or peptide comprising at least a portion of atransferrin receptor or of another brain capillary endothelial cellreceptor. The animal is maintained under conditions whereby antibodiesreactive with a transferrin receptor are produced. Blood is collectedfrom the animal upon reaching a desired titer of antibodies. The serumcontaining the polyclonal antibodies (antisera) is separated from theother blood components. The polyclonal antibody-containing serum canoptionally be further separated into fractions of particular types ofantibodies (e.g. IgG, IgM).

The ligand-neuropharmaceutical agent fusion proteins or conjugates canbe administered orally, by subcutaneous or other injection,intravenously, intra-arterially, intramuscularly, parenterally,transdermally, nasally or rectally. The form and concentration in whichthe conjugate is administered (e.g., capsule, tablet, solution,emulsion) will depend at least in part on the route by which it isadministered.

A therapeutically effective amount of a ligand-neuropharmaceutical agentfusion protein or conjugate is that amount necessary to significantlyreduce or eliminate symptoms associated with a particular neurologicaldisorder. The therapeutically effective amount will be determined on anindividual basis and will be based, at least in part, on considerationof the individuals's size, the severity of symptoms to be treated, theresult sought, the specific ligand, etc. Thus, the therapeuticallyeffective amount can be determined by one of ordinary skill in the artemploying such factors and using no more than routine experimentation.

The present invention will be illustrated by the following examples:

EXAMPLE 1 In Vitro Binding of Murine Monoclonal Antibodies to HumanBrain Endothelial Cells

Two murine monoclonal antibodies, B3/25 and T58/30, described byTrowbridge (U.S. Pat. No. 4,434,156 issued Feb. 28, 1984, and Nature294, pp. 171-173 (1981)), the contents of both are hereby incorporatedby reference, which recognize the human transferrin receptor were testedfor their ability to bind to human brain capillary endothelial cells.Hybridoma cell lines which produce B3/25 and T58/30 antibodies wereobtained from the American Type Culture Collection (ATCC) in Rockville,Md., and grown in DMEM medium supplemented with 2.0 mM glutamine, 10.0mM HEPES (pH 7.2), 100 μM non-essential amino acids and 10%heat-inactivated fetal calf serum. The hybridoma cultures were scaled-upin 225 cm² T-flasks for the production of milligram quantities of IgGantibody. The hybridoma supernatants were concentrated 50× using vacuumdialysis and applied to a protein-A sepharose column using the BioRadMAPS buffer system. Purified antibody was eluted from the column,dialyzed against 0.1 M sodium phosphate (pH 8.0), concentrated andstored in aliquots at -20° C.

Primary cultures of human brain endothelial cells were grown inflat-bottom 96-well plates until five days post-confluency. The cellswere then fixed using 3.0% buffered formalin and the plate blocked with1.0% bovine serum albumin (BSA) in Dulbecco's phosphate buffered saline(DPBS). Aliquots (100 μl) of the B3/25 or T58/30 antibodies, either inthe form of culture supernatants or purified protein, were then added tothe wells (antibody concentrations were in the range of 1-50 μg/ml).Antibody which had specifically bound to the fixed cells was detectedusing a biotin-labeled polyclonal goat-anti-mouse IgG antisera followedby a biotinylated horseradish peroxidase (HRP)/avidin mixture (AvidinBiotin Complex technique). Positive wells were determined using aTitertek Multiscan Enzyme Linked Immunosorbent Assay (ELISA) platereader. The results showed that both antibodies bind to human braincapillary endothelial cells with the T58/30 antibody exhibiting a higherlevel of binding.

These same antibodies were also tested for binding to human braincapillaries using sections of human brain tissue that were fresh frozen(without fixation), sectioned on a cryostat (section thickness was 5-20μm), placed on glass slides and fixed in acetone (10 minutes at roomtemperature). These sections were then stored at -20° C. prior to use.

The slides containing the human brain sections were allowed to come toroom temperature prior to use. The sections were then rehydrated in DPBSand incubated in methanol containing 0.3% H₂ O₂ to block endogenousperoxidate activity. The sections were blocked for fifteen minutes in asolution containing 0.2% non-fat dry milk and 0.2%methylmannopyranoside. B3/25 and T58/30 antibodies, purified asdiscussed above, were applied to the sections at a concentration of 5-50μg/ml and incubated at room temperature for one to two hours. Antibodythat specifically bound to the tissue was detected using theAvidin-Biotin Complex (ABC) technique as described above for the ELISAassay. Staining of capillaries in the human brain sections was observedwith both the B3/25 and T58/30 antibodies. The T58/30 antibody alsodisplayed some binding to the white matter of the brain cortex.

EXAMPLE 2 In-Vitro Binding of Murine Monoclonal Antibody 0X-26 to RatTransferrin Receptor

The 0X-26 murine antibody, which recognizes the rat transferrinreceptor, has been shown in vivo to bind to brain capillary endothelialcells (Jeffries et al., cited supra). The murine hybridoma line whichproduces the 0X-26 murine antibody was obtained and the hybridoma cellline was grown in RPMI 1640 medium supplemented with 2.0 mM glutamineand 10% heat-inactivated fetal calf serum. The 0X-26 antibody waspurified using the affinity chromatography technique described inExample 1.

The purified antibody was tested in vitro as described for theanti-human transferrin receptor antibodies in Example 1 to determinewhether it would bind to brain capillaries in fresh frozen,acetone-fixed rat brain sections. The results showed that the 0X-26anti-transferrin receptor antibody did bind to capillaries in rat brainsections in vitro.

EXAMPLE 3 In-Vivo Binding of 0X-26 Murine Monoclonal Antibody to RatTransferrin Receptor

Dose Range

The anti-rat.transferrin receptor antibody 0X-26 was tested in vivo byinjecting purified antibody (purification as described in Example 1)into female Sprague-Dawley rats (100-150 gm) via the tail vein. Prior toinjection, the rats were anesthetized with halothane. The samples,ranging from 2.0 mg to 0.05 mg of antibody/rat were injected into thetail vein in 400 μl aliquots. All doses were tested in duplicateanimals. One hour post-injection, the animals were sacrificed andperfused through the heart with DPBS to clear the blood from the organs.Immediately after the perfusion was completed, the brain was removed andquick frozen in liquid nitrogen. The frozen brain was then sectioned(30-50 μm) on a cryostat and the sections placed on glass microscopeslides. The brain sections were air dried at room temperature one to twohours before fixation in acetone (10 minutes at room temperature). Afterthis treatment the sections could be stored at -20° C.

The 0X-26 antibody was localized in the brain sections usingimmunohistochemistry as described above for the in vitro experiments inExample 1. The addition of the primary antibody was unnecessary in thatit is present in the brain sections. The results indicated that the0X-26 antibody binds to rat brain capillary endothelial cells and thatdoses of as little as 50 μg result in detectable levels of antibody inthe brain using the methods described herein. Doses above 0.5 mg did notappear to show significantly more antibody binding to the endothelialcells, suggesting that the sites for antibody binding may be saturated.No specific binding to capillary endothelium was detected in the liver,kidney, heart, spleen or lung.

A non-specific antibody of the same subclass as 0X-26 (IgG 2a) was alsotested in vivo to show that the binding of 0X-26 to rat brainendothelial cells that has been observed is specific to the 0X-26antibody. 0.5 mg of the control antibody was injected per rat asdescribed above. The results indicate that the staining pattern observedwith the 0X-26 antibody is specific to that antibody.

Time Course

After establishing that the 0X-26 antibody is detectable in the ratbrain capillaries after in vivo administration, the time frame in whichthis binding occurred was determined. Using 0.5 mg of purified 0X-26antibody as the standard dose, brain sections taken from animalssacrificed 5 minutes, 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours and24 hours post-injection were examined for the presence of 0X-26antibody. All doses were administered in 400 ul aliquots and each timepoint was tested in duplicate animals. Samples were injected and therats were processed at the various times postinjection as describedabove in the dose range section.

The results showed that the 0X-26 antibody can be detected in or on therat brain capillary endothelial cells as early as five minutes and aslate as 24 hours post-injection. At 4 and 8 hours post-injection, thestaining pattern of the antibody is very punctate suggesting that theantibody has accumulated in vesicular compartments either in endothelialor perivascular cells.

EXAMPLE 4 The Use of a Conjugate of 0X-26 Murine Monoclonal Antibody forTransferring Horseradish Peroxidase Across the Blood Brain Barrier

Horseradish Peroxidase (HRP; 40 kD) was chosen as a compound to bedelivered to the brain because it is similar in size to severaltherapeutic agents and it can be easily detected in the brain using anenzymatic assay. HRP was conjugated to the 0X-26 antibody using anon-cleavable periodate linkage and the ability of the antibody tofunction as a carrier of compounds to the brain was examined. Theantibody conjugate was tested in vivo to determine if the antibody coulddeliver HRP to the brain.

The antibody (10 mg) was first dialyzed overnight against 0.01 M sodiumbicarbonate (pH 9.0). The HRP (10 mg) was dissolved in 2.5 ml deionizedwater, 0.1 M sodium periodate (160 μl) was added and the mixture wasincubated for five minutes at room temperature. Ethylene glycol (250 μl)was added to the HRP solution followed by an additional five minuteincubation. This solution was then dialyzed overnight against 1.0 mMsodium acetate buffer (pH 4.4). To the dialyzed 0X-26 antibody (2.0 ml,5.08 mg/ml) was added 200 μl of 1.0 M sodium bicarbonate buffer, pH 9.5and 1.25 ml of the dialyzed HRP solution. This mixture was incubated inthe dark for two hours followed by the addition of 100 μl of 10 mg/mlsodium borohydride. The resulting mixture was incubated for twoadditional hours in the dark at 4° C. The protein was precipitated fromthe solution by the addition of an equal volume of saturated ammoniumsulfate and resuspended in a minimal volume of water. Free antibody wasremoved from the mixture by chromatography on a concanavalin A-sepharosecolumn (a column which binds HRP and the HRP-antibody conjugate andallows the free antibody to pass through). The free HRP was removed bychromatography on a protein A-sepharose column which retains theantibody-HRP conjugate. The final product had an HRP/antibody ratio of4/1.

A time course experiment identical to that described in Example 3 wasperformed using the antibody-HRP conjugate. The antibody-HRP conjugate(0.5 mg) was injected in a 400 μl aliquot/rat. The animals weresacrificed at the various times post-injection and the brains processedas described above in Example 3. The antibody HRP conjugate waslocalized in the brain either by staining for antibodyimmunohistochemically as described in Example 1 or by directly stainingthe brain sections for the presence of HRP. To detect HRP, the slideswere first allowed to come to room temperature before incubating inmethanol for thirty minutes. The brain sections were then washed in DPBSand reacted with 3,3'-diamino benzidine (DAB), the substrate for HRP.The results showed that the 0X-26 antibody HRP conjugate binds to ratbrain capillary endothelial cells in a manner identical to that of theunconjugated antibody. The punctate staining 4-8 hours after injectionwhich was seen with the antibody alone is also seen with the antibodyconjugate, suggesting that the conjugate can also be going into thepericytes on the abluminal side of the blood brain barrier. Takentogether, these results indicate that the 0X-26 antibody can deliver aprotein molecule of at least 40 KD to the brain.

EXAMPLE 5 The In-Vivo Delivery of Adriamycin to the Brain by MurineMonoclonal Antibody 0X-26

A non-cleavable linker system similar to that used in Example 4, wasused to couple the chemotherapeutic drug adriamycin to the 0X-26antibody. The availability of antibodies that can detect adriamycin aswell as the system previously described in Example 1 for detecting theantibody carrier allowed the use of immunohistochemical techniques formonitoring the localization of the antibody carrier as well as thedelivery of adriamycin to the brain.

To conjugate adriamycin to the antibody, the drug (10 mg in 0.5 ml DPBS)was oxidized by the addition of 200 μl of 0.1 M sodium periodate. Thismixture was incubated for one hour at room temperature in the dark. Thereaction was quenched by the addition of 200 μl of ethylene glycolfollowed by a five minute incubation. The 0X-26 antibody (5.0 mg in 0.5ml of carbonate buffer (pH 9.5)) was added to the oxidized adriamycinand incubated at room temperature for one hour. Sodium borohydride (100μl of 10 mg/ml) was added and the mixture was incubated for anadditional two hours at room temperature. The free adriamycin wasseparated from the 0X-26 antibody-adriamycin conjugate by chromatographyon a PD-10 column. The adriamycin/OX-26 antibody ratio within theconjugate was 2/1 for this particular batch of conjugate.

The effectiveness of the 0X-26 antibody as a carrier for deliveringadriamycin to the brain was determined by administering 0.5 mg of theantibody-adriamycin conjugate in a 400 μl aliquot per rat by injectionvia the tail vein. One hour post-injection, the rat was sacrificed andthe brain processed as described in Example 1. All injections wereperformed in duplicate. As a control, 400 μg of free adriamycin in a 400μl aliquot was also injected into a rat. Immunohistochemistry was usedto detect both the carrier 0X-26 antibody and the adriamycin in the ratbrain sections. In the case of adriamycin, polyclonal rabbitanti-adriamycin antisera was applied to the sections followed by abiotinylated goat anti-rabbit IgG antisera. This was then followed bythe addition of a biotinylated HRP/avidin mixture and enzymaticdetection of HRP.

The results indicate that both the 0X-26 antibody and the conjugatedadriamycin localized to the rat brain capillary endothelial cells afterin vivo administration. There is no evidence that free adriamycin bindsto brain capillary endothelial cells or enters the brain.

An adriamycin-OX-26 conjugate coupled via a carbodiimide linkage wasalso synthesized (drug/antibody ratio of 10/1) and tested in vivo. Theresults of this experiment were essentially identical to that obtainedwith the periodate-linked antibody-drug conjugate. In both cases,staining for the antibody carrier was quite strong and was visualized inthe capillaries in all areas of the brain. This staining was evenlydistributed along the capillaries. Staining for adriamycin was lessintense but again was seen in capillaries throughout the brain. Somepunctate staining was observed which suggests accumulation in pericyteswhich lie on the brain side of the blood-brain barrier.

EXAMPLE 6 In Vivo Delivery of Methotrexate to the Brain by MurineMonoclonal Antibody 0X-26.

A noncleavable carbodiimide linkage was used to couple methotrexate tothe 0X-26 murine monoclonal antibody. A system analogous to thatdescribed in Example 5 was used to monitor the delivery of both themethotrexate and the carrier antibody to the brain capillary endothelialcells.

Methotrexate was coupled to murine monoclonal antibody 0X-26 via itsactive ester. Briefly, 81 mg (0.178 mM) of methotrexate (Aldrich) wasstirred with 21 mg (0.182 mM) of N-hydroxysuccinimide (Aldrich) in 3 mlof dimethylformamide (DMF) at 4° C.Ethyl-3-dimethylaminopropyl-carbodiimide (180 mg,EDC;0.52 mM) was addedto this solution and the reaction mixture was stirred overnight. Thecrude ester was purified from the reaction by-products by flashchromatography over silica gel 60 (Merck) using a solution of 10%methanol in chloroform as an eluant. The purified active ester fractionswere pooled and concentrated to dryness. The ester was dissolved in 1 mlof DMF and stored at -20° C. until use. 50 mg (50%) of active ester wasrecovered as determined by A₃₇₂ (ε₃₇₂ =7200) A solution of 0X-26containing 2.1 mg (14 nmoles) of antibody in 0.9 ml of 0.1 M phosphate(pH 8.0) was thawed to 4° C. To this stirred antibody solution was added1.4 μl (140 nmoles) of the active ester prepared as described above.After 16 hours at 4° C., the mixture was chromatographed over SephadexPD-10 column (Pharmacia) using phosphate buffered saline (PBS) toseparate conjugate from free drug. The fractions containing theantibody-methotrexate conjugate were pooled. Antibody and drugconcentration were determined spectrophotometrically as described byEndo et al. (Cancer Research (1988) 48: 3330-3335). The final conjugatecontained 7 methotrexates/antibody.

The ability of the 0X-26 monoclonal antibody to deliver methotrexate tothe rat brain capillary endothelial cells was tested in vivo byinjecting 0.2 mg of conjugate (in 400μl) into each of two rats via thetail vein. The animals were sacrificed one hour post-injection and thebrains processed for immunohistochemistry as described in Example 1. Todetect methotrexate in the brain, a rabbit antisera raised againstmethotrexate was used as the primary antibody. A biotinylatedgoat-anti-rabbit antisera in conjunction with a mixture of biotinylatedHRP and avidin was then used to visualize methotrexate in the rat brain.The carrier antibody was detected as described previously.

The results of these experiments indicate that methotrexate in the formof a conjugate with 0X-26 does accumulate along or in the capillaryendothelial cells of the brain. The staining observed for methotrexateis comparable in intensity to that seen for the carrier. The stainingappears to be in all areas of the brain and is evenly distributed alongthe capillaries.

EXAMPLE 7 Antibody Derivatives

The Fc portion of the 0X-26 murine monoclonal antibody was removed todetermine whether this would alter its localization to or uptake by therat brain capillary endothelial cells. F(ab')₂ fragments of 0X-26 wereproduced from intact IgG's via digestion with pepsin. A kit availablefrom Pierce Chemical Co. contains the reagents and protocols forcleaving the antibody to obtain the fragments The F(ab')₂ fragment (0.2mg doses) in 400 μl aliquots were injected into rats via the tail vein.A time course experiment identical to that done with the intact antibody(Example 2) was then performed. F(ab')₂ fragment was detectedimmunohistochemically using a goat anti-mouse F(ab')₂ antisera followedby a biotinylated rabbit anti-goat IgG antisera. A biotinylatedHRP/avidin mixture was added and the antibody complex was visualizedusing an HRP enzymatic assay. The results indicate that the F(ab)₂fragment of the 0X-26 antibody binds to the capillary endothelial cellsof the rat brain.

EXAMPLE 8 Measurement of 0X-26 in Brain Tissue

To quantitate the amount of 0X-26 which accumulates in the brain,radioactively-labelled antibody was injected into rats via the tailvein. Antibodies were labelled with either ¹⁴ C-acetic anhydride or ³H-succinimidyl propionate essentially as described in Kummer, U.,Methods in Enzymology, 121: 670-678 (1986), Mondelaro, R. C., andRueckert, R. R., J. of Biological Chemistry, 250: 1413-1421 (1975),hereby incorporated by reference. For all experiments, the radiolabelledcompounds were injected as a 400 μl bolus into the tail vein of femaleSprague-Dawley rats (100-125 gms) under Halothane anesthesia and theanimals were sacrificed at the appropriate time post-injection using alethal dose of anesthetic. A ³ H-labelled IgG2a control antibody wasco-injected with the ¹⁴ C-labelled 0X-26 to serve as a control fornon-specific radioactivity in the brain due to residual blood. At theappropriate time post-injection, animals were sacrificed and the brainswere removed immediately and homogenized in 5 ml of 0.5% sodiumdodecylsulfate using an Omni-mixer. An aliquot of the homogenate wasincubated overnight with 2 ml of Soluene 350 tissue solubilizer prior toliquid scintillation counting. All data were collected asdisintegrations per minute (dpm). Blood samples were centrifuged topellet red blood cells (which do not display significant binding ofradiolabelled materials) and the radioactivity in an aliquot of serumdetermined using liquid scintillation counting.

The amount of antibody associated with the brain was determined atvarious times post-injection to examine the pharmacokinetics of brainuptake. In addition, the amount of labelled antibody in the blood wasmeasured so that the rate of clearance from the bloodstream could bedetermined. This information was also used to calculate the amount ofradioactivity in the brain due to blood contamination, which was thensubtracted from the total to give the amount of antibody that isspecifically associated with the brain.

A peak level of ¹⁴ C-labelled 0X-26 corresponding to approximately 0.9%of the injected dose was reached in the brain between 1 and 4 hourspost-injection as illustrated in FIG. 1 (with the values shown as meansplus or minus standard error of the mean (SEM) and N=3 rats per timepoint). The amount of radioactivity associated with the brain decreasedsteadily from 4 to 48 hours post-injection, at which point it leveledoff at approximately 0.3% of the injected dose. The accumulation of0X-26 in the brain was significantly reduced by the addition ofunlabelled monoclonal antibody (0.5 or 2.0 mg in the bolus injection).As an additional control, a ³ H-IgG2a control antibody was co-injectedwith the ¹⁴ C-0X-26. The control antibody did not accumulate in thebrain and represented the blood contamination of the brain.

In contrast to the levels in the brain, the blood level of 0X-26 droppedquite dramatically immediately after injection such that by 1 hourpost-injection, the percent of injected dose in 55 μl of blood (thevolume of blood associated with the brain) was approximately 0.16% asillustrated in FIG. 1. This corresponds to a value of approximately 20%of the injected dose in the total blood volume of the rat. Extraction oftotal IgG from serum followed by polyacrylamide gel electrophoresis(PAGE) and autoradiography did not reveal detectable levels of 0X-26degradation indicating that the antibody remains intact in the blood aslong as 48 hours after injection.

EXAMPLE 9 Distribution of 0X-26 in Brain Parenchyma and Capillaries

To demonstrate that anti-transferrin receptor antibody accumulates inthe brain parenchyma, homogenates of brains taken from animals injectedwith labelled 0X-26 were depleted of capillaries by centrifugationthrough dextran to yield a brain tissue supernatant and a capillarypellet. Capillary depletion experiments followed the procedure ofTriguero, et al., J. of Neurochemistry, 54: 1882-1888 (1990), herebyincorporated by reference. As for the brain uptake experiments ofExample 8, the radiolabelled compounds were injected as a 400 μl bolusinto the tail vein of female Sprague-Dawley rats (100-125 gm) underHalothane anesthesia and the animals were sacrificed at the appropriatetime post-injection using a lethal dose of anesthetic. A ³ H-labelledIgG 2a control antibody was co-injected with the ¹⁴ C-labelled 0X-26 toserve as a control for non-specific radioactivity in the brain due toresidual blood. After sacrifice, the brains were removed and kept onice. After an initial mincing, the brains were homogenized by hand (8-10strokes) in 3.5 ml of ice cold physiologic buffer (100 mM NaCl, 4.7 mMKCl, 2.5 mM CaCl₂, 1.2 mM KH₂ PO₄, 1.2 mM MgSO₄, 14.5 mM HEPES, 10 mMD-glucose, pH 7.4). Four ml of 26% dextran solution in buffer was addedand homogenization was continued (3 strokes). After removing an aliquotof the homogenate, the remainder was spun at 7200 rpm in a swingingbucket rotor. The resulting supernatant was carefully removed from thecapillary pellet. The entire capillary pellet and aliquots of thehomogenate and supernatant were incubated overnight with 2 ml of Soluene350 prior to liquid scintillation counting. This method removes greaterthan 90% of the vasculature from the brain homogenate (Triguero et al.,cited supra).

A comparison of the relative amounts of radioactivity in the differentbrain fractions as a function of time indicates whether transcytosis ofthe labelled antibody has occurred. The amount of 0X-26 in total brainhomogenate, the brain parenchyma fraction and the brain capillaryfraction at an early time (30 minutes) and a later time (24 hours)post-injection is illustrated in FIG. 2. The values in FIG. 2 are shownas means±SEM with N=3 rats per time point. At the 30 minute time point,more of the radiolabelled antibody is associated with the capillaryfraction than with the brain parenchyma fraction (0.36% of the injecteddose (%ID) and 0.23% ID, respectively). By 24 hours post-injection, thedistribution is reversed and the majority of the radioactivity (0.36%ID) is in the parenchymal fraction as compared to the capillary fraction(0.12 ID). The redistribution of the radiolabelled 0X-26 from thecapillary fraction to the parenchyma fraction is consistent with thetime dependent migration of the anti-transferrin receptor antibodyacross the blood-brain barrier.

EXAMPLE 10 Biodistribution and Brain Uptake of Anti-Human TransferrinReceptor Antibodies in Cynomolgous Monkeys

A collection of 32 murine monoclonal antibodies which recognize variousepitopes on the human transferrin receptor were examined for reactivitywith brain capillary endothelial cells in sections from human, monkey(cynomolgous), rat and rabbit brain samples by the immunohistochemicalmethods described in Example 1. These antibodies were obtained from Dr.Ian Trowbridge of the Salk Institute, LaJolla, Calif. All 32 antibodiesdisplayed some reactivity with human brain endothelial cells. Twoantibodies reacted very weakly with rabbit brain capillaries and nonereacted with rat. While 21 of the antibodies reacted with monkey braincapillaries, only 2 displayed strong reactivity comparable to that seenwith human brain capillaries. These 2 antibodies are herewithin referredto as 128.1 and Z35.2.

These antibodies were used to determine the tissue distribution andblood clearance of the ¹⁴ C-labelled anti-human transferrin receptorantibodies 128.1 and Z35.2 in 2 male cynomolgous monkeys. 128.1 or Z35.2was administered concurrently with a ³ H-labelled control IgG to one ofthe monkeys with an intravenous catheter. During the course of thestudy, blood samples were collected to determine the clearance of theantibodies from the circulation. At 24 hours post-injection, the animalswere euthanized and selected organs and representative tissues werecollected for the determination of isotope distribution and clearance bycombustion. In addition, samples from different regions of the brainwere processed as described for the capillary depletion experiments inExample 9 to determine whether the antibodies had crossed theblood-brain barrier. The results of the capillary depletion experimentswere performed on samples from the cortex, frontal cortex, cerebellumand striatum. All samples had greater than 90% of the 128.1 or Z35.2 inthe brain parenchyma, suggesting that the antibodies crossed theblood-brain barrier. The levels of the control antibody in the samesamples were from 5 to 10-fold lower. Using the average brain homogenatevalue for dpm/G tissue, the percent injected dose of 128.1 in the wholebrain is approximately 0.2-0.3%. This compares to a value of 0.3-0.5%for 0X-26 in the rat at 24 hours post-injection. A comparison of theratios of 128.1 to the control antibody for various organs isillustrated in FIG. 3. Similar results were obtained for Z35.2. Theseresults suggest that 128.1 is preferentially taken up by the brain ascompared to control antibody. For the majority of organs and tissuestested, the ratio of 128.1 to control is less than 2.

EXAMPLE 11 Construction of a NGF-IgG3 Hinge-Transferrin Fusion Gene,Expression of the NGF-IgG3 Hinge-Transferrin Fusion Gene as a FusionProtein and Assay of the Fusion Protein Constituents

A fusion protein comprised of human NGF and human transferrin wasconstructed using the human IgG3 hinge region as a linker between theNGF and transferrin polypeptides. The IgG3 hinge region, which is about60 amino acids long and includes a number of cysteine residues, waschosen as a connector between the NGF and transferrin polypeptides. Thepotential for disulfide formation within the immunoglobulin hinge regionwas envisioned as increasing the probability of allowing thedimerization of the NGF polypeptides into a more native NGFconfiguration.

Preparation of a Bacterial Vector for Insertion of the NGF Gene.

Bacterial plasmid pAT3442 (FIG. 4) was used as the starting plasmid forconstruction of the NGF-hinge-transferrin gene fusion. It containedhuman genomic DNA encoding the IgG3 CH1 (constant region 1 of the heavychain) and hinge region and the human transferrin cDNA. This plasmid wasconstructed by the following procedure.

Bacterial plasmid pAT3442 was derived from vector PAT153 ("PracticalGuide to Molecular Cloning, 1984, Bernard Perbal, John Wiley Publisher).pAT153 was first modified to remove the EcoRI site by cleavage of thisvector with EcoRI filling in of the 5' overhang regions with the use ofDNA polymerase, and religation. This derivative was designated pAT153.7.

A SalI-BamHI fragment containing the sequence coding for human IgG3constant region and the hinge region with its associated introns wasisolated from phage lambda libraries as described in Dangl, J. L., (1986Dissertation, Stanford University, Stanford, Calif.). A large portion ofthe untranslated region was eliminated from the 3' end of the IgG3 geneby cleavage with PvuII (which cleaves multiple times within this region)and religation. This fragment was further modified by site directedmutagenesis to contain a PvuII site at the 5' end of the CH2 region;cleavage with SmaI and introduction of an EcoRI linker resulted in theSmaI site in the CH3 region being joined to the SmaI site 0.6 kbupstream of the BamHI site with an EcoRI site separating them. ThisSalI-BamHI fragment with the PvuII and EcoRI sites was cloned into SalIand BamHI cleaved pAT153.7 (pAT153 with its EcoRI site deleted byfilling in; also called pAT3404) yielding pAT3408.

A 2.4 kb Pst I fragment containing the human transferrin cDNA sequencewas isolated from clone Tf (U.S. Pat. No. 5,026,651) and cloned into thePstI site of pBluescript II KS [Stratagene] creating pKS3436. A PvuIIsite was introduced at the 3' end of the leader sequence of thetransferrin by standard site-directed mutagenesis procedures,thereby-creating pKS3438. An EcoRI site beyond the 3' end of thetransferrin gene and the newly introduced PvuII site were used to clonethe 2.4 kb fragment containing the transferrin coding sequence with itsassociated polyA site into pAT3408; transferrin thereby replaced the CH2and CH3 domains of IgG3. As a consequence of this manipulation, anucleotide sequence encoding the amino acid sequence ala-ala precedesthe mature transferrin coding sequence. Approximately 600 bp of theregion 3' of IgG3 adjacent to the BamHI site were adjacent to the 3' endof the transferrin gene. The resulting plasmid was designated pAT3442(FIG. 4).

The unique Eco RV site downstream of the 3' untranslated IgG3 sequencesin plasmid pAT3442 was converted to an XbaI site by digestion with EcoRVand ligation of a synthetic linker containing an XbaI restriction sitein order to facilitate future cloning into appropriate mammalianvectors. A clone containing the new XbaI site was designated PATX. A mapof the CH1-hinge-transferrin (CH1-hinge-Tf) region of pATX is shown inFIG. 5A.

The CH1 coding sequence in pATX was then replaced with the NGF gene.pATX was modified using polymerase chain reaction (PCR) techniques intwo steps. The 5' PCR primer, HTF-1 (shown below), contained SalI andXhoI cloning sites near its 5' end, and 14 bases complementary to thefirst intron of the hinge region at its 3' end.

    (SEQ.ID.NO.1)                                                                                 SalI   XhoI    first hinge intron                                HTF-1  5'-GG GTCGAC CTCGAG GGT GAG AGG CCA GC-3'                       

The 3' primer (HTF-2) was complementary to a sequence within the firstintron of the hinge region, approximately 400 nucleotides downstream ofthe 5' primer, and included a BglII cloning site.

    (SEQ.ID.NO.2)                                                                        first hinge intron BglII                                                 HTF-2    5'-GGAGTTACTC AGATCT GGGAAG-3'                                 

The primers were combined with pATX template DNA and, following the PCRprocedures, the 400 bp amplified fragment was gel purified, digestedwith SalI and BglII, and cloned into pATX which had been digested tocompletion with SalI and partially with BglII (to cleave at one of twoBglII sites of this plasmid). The ligated sample was transformed intoE.coli DH1 cells and a clone having the 400 bp ligated fragment in placeof the CH1 region was identified by restriction digestion analysis. Thisclone was designated pATXX; a map of the hinge-transferrin region ofpATXX is shown in FIG. 5B.

Isolation and Cloning of the NGF gene.

The pre-pro form of the NGF gene, which is about twice the size of themature β-NGF coding sequence and contains the signals for proteinsecretion and protein folding, was amplified by PCR techniques fromhuman erythrocyte genomic DNA (purchased from Clontech, Palo Alto,Calif.) using the following primers.

                          PNGF 1 (5' Primer)               (SEQ.ID.NO.3)                                                                       HindIII Xhol                                                             Xbal                                                                              Start                                                                     5'-G AAGCTT                                                                  CTCGAG TCTAGA                                                                 CCAGGTGCATAGCGTA                                                              ATG TCC-3'                                                                      - PNGF 2 (3'                                                                Primer) (SEQ.ID.NO.                                                           4)                             SalI   Xhol                                                             5'-C GTCGAC CTCGAG TCTCACAGCCTTCCTGCTGAGC-3'                            

PNGF1 and PNGF2 were partially homologous to sequences at the 5' and 3'ends of the pre-pro NGF gene, respectively, and were devised to createXhol cloning sites at either end of the NGF gene. The 5' primeradditionally contained an Xbal cloning site.

Using the PNGF1 and PNGF2 primers, a fragment of approximately 800 bpcontaining the pre-pro NGF gene was amplified. The resulting DNA wasdigested with Xhol, and the (approximately) 800 bp fragment was gelpurified and ligated into Xhol digested pATXX. A clone designatedpATXXNGF was identified which had the pre-pro NGF gene inserted upstreamand adjacent to the IgG3 hinge region in the same orientation as thetransferrin gene thereby creating an NGF-IgG3 hinge-transferrin (NHT)gene fusion. A map of this region of pATXXNGF is shown in FIG. 5C. (TheXhoI site 3' to the pre-pro NGF coding region of pATXXNGF encodes aleu-glu which precedes the IgG3 hinge region).

A partial DNA sequence of the junctions between the NGF-hinge andhinge-transferrin sequences were determined in order to verify thesequence of the primers and to confirm the correct reading frame withinthe newly formed gene fusion. The determined sequences revealed onenucleotide change, a G to A transition, which resulted in an arginine toglutamine change at amino acid position 80 in the pre-pro portion ofNGF.

Reconstruction of the NGF-Hinge-Transferrin Genetic Fusion in MammalianExpression Vectors.

The NGF-IgG3 hinge-transferrin gene fusion was cloned stepwise intomammalian expression vector pcDNAI/AMP (Invitrogen) for transfectioninto COS cells (ATCC Accession Number CRL 1651) using the followingprocedure.

The pre-pro NGF portion of the gene fusion was first amplified frompATXXNGF using PCR techniques. The 5' PCR primer, PNGF1, was describedabove and the 3' PCR primer, PNGF3, is shown below.

    (SEQ.ID.NO.5)                                                                   PNGF3 (3' Primer)                                                                  SalI    Xbal                                                             5'-C GTCGAC TCTAGA TTA TCTCACAGCCTTC-3'                                                          Stop                                                 

Using the PNGF1 and PNGF3 primers, an approximately 800 bp fragmentcontaining the pre-pro NGF gene was amplified from pATXXNGF. Theresulting DNA was digested with Xbal, ligated into Xbal digestedbacterial vector pUC18 (Boehringer-Mannheim) and transformed intocompetent E.coli TOP10F cells (Invitrogen). Clones containing the NGFgene in either orientation were identified by restriction analysis anddesignated pUCNGF1 (clockwise orientation) and pUCNGF2 (counterclockwiseorientation) (FIG. 6).

The entire NHT gene fusion was then recreated in pcDNAI/Amp using athree-part ligation. The first fragment, an approximately 600 bpfragment containing most of the NGF gene, was removed from pUCNGF2 bydigestion with BamHI, which cleaves within the polylinker sequenceupstream of the pre-pro NGF gene sequence, and Scal, which cleaves nearthe 3' end of the NGF gene. The second fragment was a 4.7 kb Scal toXbal fragment isolated from pATXXNGF which contained the remainder ofthe 3' end of the NGF gene, the hinge region and the entire transferringene. The third fragment was the pcDNAI/Amp vector which had beendigested within its polylinker sequence with BamHI and Xbal and gelpurified. The three fragments were ligated together (FIG. 7) andtransformed into E.coli cells. A plasmid containing all three fragmentswas identified and designated pcDNAI/AmpNHT. The DNA sequence was againdetermined to verify the NGF and transferrin coding sequences. Thedetermined sequence revealed a T to C transition in NGF which resultedin a valine to alanine change at amino acid 35, and three changes fromthe published transferrin nucleotide sequence that did not result inamino acid changes.

Assay for NGF and Transferrin in the Fusion Protein Expressed inMammalian Cells.

Expression plasmid pcDNAI/AmpNHT was transfected and transientlyexpressed in COS cells. The fusion protein in culture supernatants wasdetected using anti-NGF antibodies, anti-transferrin antibodies, orpurified transferrin receptor by standard ELISA procedures.

Briefly, capture antibody (anti-NGF or anti-transferrin), which wasspecific for either the NGF or transferrin portions of the fusionprotein, was coated in the wells of a 96-well plate. The wells werewashed (PBS-0.05% Tween), blocked with 1% bovine serum albumin (BSA),and supernatants from transfected COS cells were added to the wells(typically in serial four-fold dilutions) and incubated for one hour atroom temperature.

A detection antibody was chosen which would recognize the other portionof the fusion protein (either anti-Tf or anti-NGF) and was added to thewells on top of the fusion protein. Bound antibody was detected afteramplification of the signals by an avidin-biotin reaction using theVectastain ABC kit (VectorLabs). Protein was quantitated byextrapolation from standard curves generated for known concentrations ofNGF or transferrin.

Alternative ELISA procedures may be used to detect and quantitate thefusion proteins. For example, the capture antibody and detectionantibody may recognize the same portion of the fusion protein.

Optimization of Expression of the NHT Fusion Proteins

In order to increase the level of expression of the NHT fusion protein,the translation initiation sequence immediately preceding the AUG startcodon of the pre-pro NGF gene was modified using PCR techniques toincorporate a Kozak consensus sequence (Kozak, 1987, Nucl. Acid. Res.15, 8125).

Two PCR primers were designed which were complementary to regionsflanking the pre-pro NGF gene sequence. The 5' primer, p45.1, containedan Smal restriction site as well as the sequence CCACC, the Kozaksequence, which has been shown to be important for efficient translationin mammalian cells, immediately preceding the ATG initiation codon ofthe pre-pro NGF gene.

                             p45.1                                                         Smal    XbaI          Kozak (SEQ.ID.NO.6)                              5'-TCC CCCGGG TCTAGA CCAGGTGCAT CCACC ATGTCCATGTTGTTC-3'                

The 3' primer, p16.1, was complementary to a sequence 3' to both the NGFcoding sequence and the BamHI site in pUCNGF1.

    p16.1                                                                            - 5'-AACAGCTATGACCATG-3' (SEQ.ID.NO.7)                                 

By PCR techniques, the pre-pro NGF coding sequence, preceded by theKozak translation initiation consensus sequence in p45.1, was amplifiedfrom pUCNGF1 using the p45.l and p16.1 primers. The resulting DNA can becloned into the mammalian expression vector, CD5lneg1.

One way to accomplish this cloning is to first insert the pre-pro NGFgene sequence into a bacterial vector, for example pGEM-2 (Promega),having compatible restriction sites for subsequent cloning into themammalian expression vector. The amplified pre-pro NGF gene can bedigested with SmaI and BamHI and ligated into the polylinker of pGEM-2between HincII and BamHI sites (since SmaI and HincII produceblunt-ended fragments) thereby producing a clone designated pGEM-2/KNGF.

The NHT fusion is then recreated by a three-part ligation as follows. AHindIII-ScaI fragment from the pGEM-2/KNGF clone (containing most of theNGF coding region) is mixed with the ScaI-XbaI fragment frompcDNAI/AmpNHT (containing the C-terminal-most portion of the NGF gene,the IgG3 hinge and the transferrin coding sequence) and HindIII+XbaIdigested pGEM-2 vector. The resulting vector has the entire NHT fusionbetween flanking XbaI sites (the 5' XbaI site from linker p45.1). ThisXbaI fragment is then cloned into the XbaI digested CD5lneg1.

CD5lneg1 contains a CMV promoter sequence, a CD5 leader sequence forsecretion of a cloned protein, a sequence encoding the hinge region andFc portion of IgG1, and a polyadenylation signal sequence (FIG. 8).

CD5lneg1 (gift of Brian Seed, Massachusetts General Hospital) can bederived from plasmid pCDM8 (Invitrogen) by deletion of the 590 bp DraIfragment within the SV40 intron sequence, the BamHI-SfiI fragmentcontaining the polyoma origin of replication, and a 20 bp NheI-SspIfragment at the 3' end of M13 ori which was removed in order toeliminate the NheI site. In addition, CD5lneg1 also contains the CD5leader sequence (Genbank reference number X04391) adjacent to the IgG1hinge-Fc region (Genbank reference number J00228) inserted between theXhoI (within polylinker) and EagI (approximately 43 bp beyondpolylinker) sites of pCDM8. The DNA sequence of the IgG1 hinge regionbetween the XhoI and EagI sites is shown in FIG. 9A-9B (Seq.I.D.NO.8).The CDS leader sequence and IgG1 exons are indicated on this figure. Theclone resulting from the insertion of the entire NHT fusion intoCD5lneg1 is designated CD5KNHT.

Vector CD5KNHT was transfected into CHO cells and expression of thefusion protein was assayed by ELISA as described above. Proteinscontaining both the transferrin and NGF sequences were expressed anddetected in the culture supernatant.

Purification of NGF-Hinge-Transferrin Fusion Protein from COS CellSupernatants.

NGF-hinge-transferrin fusion protein present in the supernatant oftransfected COS cells was purified using an anti-NGF affinity column.Medium from cells transfected with CD5KNHT was loaded overnight bygravity onto a

Sepharose column to which rat anti-mouse NGF monoclonal antibody hadbeen bound (e.g., antibody 1G3, Saffron et al., Brain Res. 1989,492:245-254). This column had been pre-equilibrated with PBS. Thecolumns were washed five times with 2 ml PBS, and NGF containingproteins were eluted with 10 ml of a 0.1 M glycine, 0.15 M NaClsolution, pH 3.0. The elution was accomplished a s five 2 ml fractionswhich were placed directly into tubes containing 50 μl lM NH₄ HCO₃ and 8μg/ml FeNH₄ citrate. The OD₂₈₀ of each of the fractions was thendetermined.

The affinity purified fractions contained a major band at MW 100 kD on areducing SDS-polyacrylamide gel. The band was recognized byanti-transferrin antibody and anti-NGF antibody.

In vitro Competition Assay for Transferrin Recelptor Binding Activity

A critical attribute of the NHT fusion protein is its ability to bind tothe transferrin receptor. Assays were performed to measure the affinityof the NHT fusion protein for the human transferrin receptor by theability of the fusion protein to compete with native transferrin forbinding to the transferrin receptor.

Transferrin receptor was purified from freshly obtained full term humanplacentas using the procedure of Turkewitz et al. (1988, J. Biol. Chem.,263: 8318-8325). Briefly, placental membranes were isolated and storedfrozen at -80° C. Frozen membranes were thawed and extracted withdetergent. Endogenous iron was chelated, greatly lowering the affinityof endogenous transferrin for the receptor. The transferrin receptor wasthen purified on a human transferrin-Sepharose column. By gel analysis,the resulting receptor had the expected molecular weight, and thepurity, as determined by scanning densitometry of the gel, was 98%. Thepurified transferrin receptor was immunoblotted using antibody 128.1 andanother commercially obtained anti-human transferrin receptor antibody(Amersham RPN.511).

For the competition assay, microtiter wells were coated with 100 μl/wellof 0.7 μg/ml purified human placental transferrin receptor in coatingbuffer (10 mM sodium carbonate, pH 9.5) overnight at 4° C. Nonspecificbinding was blocked by incubation with 200 μl/well of 1% w/v bovineserum albumin (BSA) for 1 hour at 37° C. The wells were then washed, and100 μl of a mixture of 4 nM ¹²⁵ I-transferrin (New England Nuclear) andvarying concentrations of competitor, either purified recombinant humantransferrin or NHT fusion protein, were applied to each well andincubated for 1 hour at room temperature. After washing, the wells wereindividually counted using a gamma counter. The percent signal remainingversus competitor concentration was plotted and the point of 50%remaining signal was determined (FIG. 10). The results demonstrated thatthe NGF-hinge-transferrin fusion protein binds to the human transferrinreceptor but has reduced affinity for this receptor compared to nativehuman transferrin.

In vitro Assay for NGF Activity.

A clonal line of rat pheochromocytoma cells (designated PC12) undergoescessation of cell division and extensive outgrowth of neurite-likeprocesses in the presence of NGF in vitro. This cell-based bioassay wasused to assess whether the expressed NHT fusion protein has NGF activityby measuring its ability to stimulate neurite outgrowth.

PC12 cells were grown in RPMI 1640 medium (Bio Whittaker) containing 5%fetal calf serum, 10% horse serum and 2 mM L-glutamine, in T75 flasksunder 5% CO₂. Ninety-six well plates were coated with 0.5 μg/cm² bovinecollagen IV at 50 μl/well, air dried overnight and exposed to UV lightfor 20 minutes prior to use. Five ml of PC12 cells were removed fromeach flask and forced through a 21 g needle about 5-10 times to break upclumps. This procedure caused the cells to lose their neurites. Thecells were diluted with media to approximately 2×10⁴ cells/ml, 50 μlwere added to each well of the collagen-coated plates (1000 cells/well),and incubated for 1-2 hours to allow the cells to attach.

Samples to be tested were filter sterilized before use. To generate adose-response curve, the samples were serially diluted in two-foldincrements in growth medium and 50 μl samples were added to the wells.Purified mouse NGF was serially diluted and plated in the same manner togenerate a standard curve. After 5 days exposure to the NGF-containingsamples, plates were scored for the presence or absence of NGF activityby counting the total number of cells and the number of cells sproutingat least one neurite that is longer than twice the diameter of the cellbody in two or three representative fields of view. The results wereexpressed as the percent of cells extending neurites as a function ofNGF concentration (FIG. 11). Panel A is the standard curve for purifiedmouse NGF. Panel B is the activity curve for the NHT fusion protein. Theresults demonstrated that the fusion protein fully retained NGFbiological activity in vitro.

EXAMPLE 12 Construction of Other NGF-Transferrin Fusion Genes

Three additional fusions of NGF and transferrin were created andtested: 1) a direct fusion of the pre-pro NGF gene and transferrin cDNA,2)a fusion of the pre-pro NGF gene and transferrin cDNA separated by asequence encoding five glycine residues (NGF-(gly)₅ -transferrin), and3) a fusion of the pre-pro NGF gene and transferrin cDNA separated by asequence encoding leu-glu (NGF-leu-glu-transferrin). The NGF-IgG3hinge-transferrin containing vector, CD5KNHT, was employed as the sourceof genetic material for creating these fusions.

Two-step PCR reactions were performed on CD5KNHT to generate theNGF-transferrin direct fusion and the NGF-(gly)₅ -transferrin fusion.For NGF-leu glu-transferrin, a one-step PCR reaction was sufficient togenerate the fragment of interest. Methods for constructing each ofthese gene fusions are described below.

Construction of the NGF-Tf Direct Fusion

The following PCR primers were used:

    P202                                                                             5'-AAGGAGGTGATGGTGTTGGGA-3'                                                                              (SEQ.ID.NO.9)                                      -     5'end of transferrin/3' end of NGF                                     P205 5'-CTCACAGTTTTATCAGGGAC TCTCACAGCCTTCCTG (SEQ.ID.NO.10)                   CTGAGC-3'                                                                     -           3' end of NGF/5' end of transferrin                              P204 5'-CAGCAGGAAGGCTGTGAGA GTCCCTGATAAAACTGT (SEQ.ID.NO.11)                   GAGATG-3'                                                                     - P203 5'-GTGTGGCAGGACTTCTTGCCT-3' (SEQ.ID.NO.12)                      

P202 and P205 were primers used to amplify the 3' two-thirds of the NGFgene. P202 was complementary to a sequence within the NGF gene 5' to theunique ScaI site. P205 was complementary to 22 bases at the 3' end ofthe NGF gene, and 20 bases at the 5' end of the transferrin gene.

Primers P204 and P203 were used to amplify the transferrin gene. P204was complementary to a 23 base sequence at the 5' end of the transferringene and 19 bases at the 3' end of the NGF gene. P203 was complementaryto a sequence within the transferrin coding sequence 3' to a uniqueBamHI site.

pcDNAI/AmpNHT DNA was mixed separately with the two sets of primers. Asa result of PCR, two fragments were amplified having overlappingprotrusions creating the junction between the NGF and transferrin codingsequences. The two fragments were gel purified and an equimolar amountof each fragment was combined for a second PCR amplification withprimers P202 and P203. The resulting product was digested with ScaI andBamHI and exchanged with the comparable ScaI to BamHI fragment ofCD5KNHT DNA that included the 3' end of the NGF gene, the hinge regionand the 5' end of transferrin coding sequence. (The BamHI digestion ofCD5KNHT was a partial digestion due to the presence of a second BamHIsite downstream of the transferrin gene.) The resulting plasmid, CD5KNT,contained a direct fusion of the NGF and transferrin genes.

Construction of the NGF-(Gly)₅ -Transferrin Fusion

The following PCR primers were used: P202 (SEQ.ID. NO. 9), P200, P201and P203 (SEQ.ID.NO.12).

                3' end of NGF/ Gly.sub.5          /                                 P200 5'-CAGCAGGAAGGCTGTGAGA GGGGGAGGTGGAGGG (SEQ.ID.NO.13)                     5' end of transferrin                                                         GTCCCTGATAAAACTGTGAGATG-3'                                                    -    5' end of transferrin/  Gly.sub.5         /                             P201 5'-CTCACAGTTTTATCAGGGAC CCCTCCACCTCCCCC T (SEQ.ID.NO.14)                  3' end of NGF                                                                 CTCACAGCCTTC-3'                                                        

Primer P200 was in part complementary to the 3' end of the NGF gene andcontained a non-complementary region encoding five glycine residuesfollowed by 23 bases complementary to the 5' end of the transferrincoding sequence. Primer P201 was complementary to the 5' end of thetransferrin, preceded by a non-complementary region encoding fiveglycine residues and the 3' end of the NGF gene.

pcDNAI/AmpNHT DNA was mixed separately with the two sets of primers(P202 and P201 were used to amplify the 3' two-thirds of the NGF geneand P200 and P203 were used to amplify the 5' end of the transferringene). Two fragments were generated having overlapping protrusions. Thetwo fragments were gel purified and an equimolar amount of each fragmentwas combined for a second PCR amplification with primers P202 and P203.The resulting amplified product was digested with ScaI and BamHI andexchanged for the comparable ScaI to BamHI fragment of CD5KNHT DNA asdescribed above. The resulting plasmid, CD5KNGT, contained a fusion ofthe NGF and transferrin coding sequences separated by a sequenceencoding five glycine residues.

Construction of the NGF-leu glu-Transferrin Fusion

The following primers were used: P206 and P203 (SEQ. ID.NO.12).

                                           XhoI  /5' end of transferrin                                                                  P206 5                                                                       '-GGAACGGC CTCGAG                                                             GTCCCTGATAAAACTGTGAG                                                          A-3' (SEQ.ID.NO.15) 

Primer P206 contained an XhoI site (CTCGAG which encodes Leu-Glu)followed immediately by a 21 base sequence complementary to the 5' endof transferrin coding sequence. Primer P203 is complementary to theregion 3' to the BamHI site within the transferrin gene. pcDNAI/AmpNHTDNA was combined with the two primers for PCR amplification. Theresulting fragment was digested with Xhol and BamHI and ligated withCD5KNHT that had been digested to completion with Xhol and partiallywith BamHI, resulting in the deletion of the IgG3 hinge region andinclusion of a leu-glu coding sequence. The resulting plasmid, CD5KNXT,contained a fusion of the NGF and transferrin coding sequence separatedby a sequence encoding leu-glu.

The NGF-transferrin direct fusion, NGF-(Gly)₅ -transferrin fusion, andNGF-leu glu-transferrin fusion were each transfected and transientlyexpressed in COS cells as in Example 11. Each of the expressed fusionproteins were detected by using anti-NGF antibodies, anti-transferrinantibodies or purified transferrin receptors. This demonstrates thatthese fusion proteins have NGF and transferrin binding characteristicssimilar to that of the native unfused proteins.

These three fusion proteins were also subjected to the PC12 neuriteoutgrowth bioassay for NGF activity as described in Example 11. Each ofthe three fusion proteins exhibited NGF biological activity asexemplified by their ability to stimulate neurite outgrowth.

EXAMPLE 13 Construction of a CNTF-Transferrin Fusion Gene

Isolation and Cloning of CNTF Gene

The human CNTF-gene was cloned from genomic DNA prepared from Jurketcells using PCR techniques. To obtain the CNTF gene sequenceuninterrupted by intron sequences, PCR was performed in two steps usingthe following four primers.

                  NdeI                                                              P31.1 5'-AGTTAA CATATG GCTTTTACTGAGCATTCAC-3' (SEQ.ID.NO.16)                   - P42.1 5'CAGGCCCTGATGCTTCACATAGGATTCCGTAAGAGC (SEQ.ID.NO.17)                 AGT-3'                                                                        - P35.1 5'-TACGGAATCCTATGTGAAGCATCAGGGCCTGA ACA-3' (SEG.ID.NO.18)                                          -           XhoI                                P42.2 5'-GGGCC CTCGAG GGACTAACTGCTACATTTTCTTGTT (SEQ.ID.NO.19)                 GTT AGC-3'                                                             

P31.1 and P42.1 were the 5' and 3' primers for amplifying the 5' exon ofCNTF. P31.1 was complementary to the 5' end of the 5' exon and containedan NdeI cloning site. P42.1 contained 21 bases complementary to the 3'end of the 5' exon and 18 bases complementary to the 5' end of the 3'exon.

P35.1 and P42.2 were the 5' and 3' primers for amplification of the 3'exon of CNTF. P35.1 was complementary to the 3' exon and contained 13additional bases which were complementary to the 3' end of the 5' exon.P42.2 was complementary to the 3' end of the 3' exon and contained anXhoI cloning site.

Genomic DNA was mixed separately with the two sets of primers. A 120 bpfragment was amplified with primers P31.1 and P42.1, and a 480 bpfragment was amplified with primers P35.1 and P42.2. The two fragmentshad a total of 31 base pairs of overlapping sequence.

In the second PCR reaction, a 600 bp fragment was amplified using amixture of gel purified 120 bp and 480 bp fragments as templatestogether with primers P31.1 and P42.2 (described above). The amplifiedfragment was digested with NdeI and XhoI and gel purified.

The NdeI-XhoI fragment was cloned into E. coli expression vector pET17xB(Novagen) that had been gel isolated after digestion with NdeI and XhoI.The ligated product was transformed into competent E. coli MC1061 cells.A clone containing the CNTF gene fragment was identified by restrictiondigestion and DNA sequence analysis and designated J6.

Cloning of Human CNTF Coding Sequence into a Mammalian Expression Vector

The human CNTF coding sequence was cloned into the CD5lneg1 mammalianexpression vector of Example 12. By the methods described below, theCNTF gene was cloned into the CD5lneg1 expression vector, adjacent tothe CD5 leader sequence, in place of the IgG1 sequences.

To prepare CNTF DNA having terminal restriction sites compatible withsites in CD5lneg1, a DNA fragment was generated by PCR techniques usingclone J6 as a template and P33.1 and P35.2 as 5' and 3' PCR primers,respectively. P33.1 was complementary to the 5' end of the CNTF gene andcontained a BlnI restriction site rather than the NdeI site. P35.2 was,in part, complementary to the 3' end of the CNTF gene and contained anEagI restriction site rather than the XhoI site.

                                        BlnI                                        P33.1 5'-CGCGGG CCTAGG CGCTTTCACAGAGCATTCACC-3' (SEQ.ID.NO.20)                 -            EagI                                                            P35.2 5'CGCGGGG CGGCCG CTTTACATTTTCTTGTTGTTGTTAG-3' (SEQ.ID.NO.21)      

Following PCR, the resulting amplified DNA fragment was treated withBlnI and EagI and ligated with the NheI and EagI digested CD5lneg1vector backbone (NheI and BlnI generate compatable protrusions; neithersite is regenerated after ligation). The ligated mixture was transformedinto competent E. coli MC1061 cells. A resulting clone having the CNTFgene preceeded by the CMV promoter and CD5 leader was identified byrestriction digestion and designated D1.

In order to minimize the possibility that the CNTF gene present inplasmid D1 contained mutations resulting from PCR procedures, NheI andBamHI, each of which cleaves once within CNTF, were used to isolate afragment containing most of the CNTF coding sequence from D1 andexchanged for the equivalent NheI-BamHI fragment of CNTF from J6. Theresulting plasmid was designated d1 (see FIG. 12A).

Construction of CNTF-Transferrin Gene Fusions

Three different gene fusions encoding CNTF-transferrin fusion proteinswere constructed. In each case, the proteins were connected by differentlinking sequences. In the first of these constructs, CNTF andtransferrin were connected by the hinge region of IgG3 (CNTF-IgG3hinge-transferrin). In the second construct, the CNTF and thetransferrin sequences were joined without an intervening linker(CNTF-transferrin). In the third construct, a sequence coding forpenta-glycine was inserted between the CNTF and transferrin genes(CNTF-(Gly)₅ -transferrin). Methods for constructing each of these genefusions are described below.

A. CNTF-IgG3 Hinge-Transferrin

The gene fusion coding for CNTF-IgG3 hinge-transferrin was constructedfor-expression in the CD5lneg1 expression vector using the followingmulti-step procedure. First, the transferrin gene was isolated andcloned into CD5lneg1 in place of the IgGl sequences. To do this, afragment containing the transferrin gene was generated by PCR techniquesusing pcDNAI/AmpNHT of Example 11 as the template and oligonucleotidesP33.2 and P36.1 as 5' and 3' PCR primers, respectively. P33.2 wascomplementary to the 5' end of the transferrin gene and contained a BlnIsite. P36.1 was complementary to the 3' end of the gene and contained anEagI site.

                    BlnI                                                            P33.2 5'-GCTTCCGT CCTAGG GGTCCCTGATAAAACTGTG-3' (SEQ.ID.NO.22)                 -             EagI                                                           P36.1 5'-CGCGGGG CGGCCG CTTTAAGGTCTACGGAAAGTGCA-3' (SEQ.ID.NO.23)       

Following PCR, the amplified fragment was digested with BlnI and EagIand cloned into the NheI and EagI digested and gel purified CD5lneg1backbone. The resulting plasmid, having the transferrin gene insertedadjacent to and downstream of the CD5 leader in place of the IgG1hinge-Fc, was designated C4. As before, in order to eliminate thepossibility that errors may have been incorporated during PCRamplification, a BamHI to Asp718 fragment of C4, which contained most ofthe transferrin gene sequence, was replaced with the equivalentBamHI-Asp718 fragment of the starting plasmid pcDNAI/AmpNHT. Theresulting plasmid was designated C*. (See FIG. 12B).

The IgG3 hinge region sequence was then inserted upstream of thetransferrin coding sequence in C4 as follows. The Xhol-Asp718 fragmentcontaining the IgG3 hinge and the 5' portion of the transferrin codingsequence (with the ala-ala coding sequence immediately upstream of thetransferrin coding sequence) from pcDNAI/AmpNHT was isolated and clonedinto the XhoI and Asp718 digested C4 backbone to generate C4-NHT. (SeeFIG. 12C).

Next, a gene fusion consisting of the CNTF gene and the transferrin genewas created in the CD5lneg1 backbone. First, a 0.7 kb XhoI-BamHIfragment from clone dl (containing the CD5 leader and most of the CNTFgene except for the 3' end) and a 1.8 kb BamHI-EagI fragment from C4-NHT(containing most of the transferrin coding sequences except for the 5'end) were ligated with XhoI and EagI digested and gel-purified CD5lneg1backbone to generate g*. (See FIG. 12D).

Then, a fragment containing the IgG3 hinge region was inserted betweenthe CNTF and transferrin genes. In order to accomplish this, an XhoIsite was created at the 3' end of the CNTF gene to make it compatiblewith the XhoI site at the 5' end of the hinge region sequence. (ThisXhoI site encoded a leu-glu 5' to the IgG3 hinge region). A new CNTFcontaining fragment was generated by PCR techniques using J6 as thetemplate and primers P33.1 (above) and P36.5 (containing an XhoI site).

    (SEQ.ID.NO.24)                                                                                         XhoI                                                   P36.5 5'TGGCCTCTCACC CTCGAG CATTTTCTTGTTGTT                                    AGC-3'                                                                 

The PCR product was digested with BlnI and XhoI and cloned into SpeI(compatible with BlnI) and XhoI digested C4-NHT to generate H45. H45contained the CNTF gene joined to the hinge coding sequence which was inturn joined to the transferrin gene. (See FIG. 12E).

Finally, a 1.5 kb BamHI fragment from H45, which contained the 3' end ofthe CNTF gene, the hinge coding sequence and the 5' end of thetransferrin gene, was isolated and cloned into BamHI digested g* togenerate plasmid gH, a plasmid containing the CNTF-hinge-transferringene fusion downstream of the CMV promoter and the CD5 leader sequence.(See FIG. 12F).

B. CNTF-Transferrin Direct Fusion

The CNTF-transferrin direct fusion was made by a two step PCR procedure.In the first step, with J6 as the template, the CNTF coding sequence wasamplified using primers P33.1 (SEQ.ID.NO.20, described above) and P38.1(below), and the transferrin gene was amplified using primers P36.2(below), P36.1 (SEQ. ID. NO. 23 described above) and templatepcDNAI/AmpNHT. P38.1 was complementary to the 5' end of the transferringene and the 3' end of the CNTF coding sequence. P36.2 was complementaryto the 3' end of the CNTF coding sequence and the 5' end of thetransferrin gene.

                          P38.1                                                                               5' end of transferrin/3' end of transferrin                                                               5                                                                           '-CTCACAGTTTTATCAGGG                                                          AC CATTTTCTTGTTGTTAG                                                          C-3' (SEQ.ID.NO.25)        - P36.2       3' end of CNTF / 5' end of transferrin                          5'-GCTAACAACAAGAAAATG GTCCCTGATAAAACTGTG-3' (SEQ.ID.NO.26)             

The two amplified fragments were gel purified and equimolar amounts werecombined for the second PCR using primers P33.1 and P36.1. The product,an intact fragment containing a CNTF-transferrin fusion gene, wasdigested with BlnI and EagI and ligated with NheI and EagI digestedCD5lneg1. A clone was identified by restriction analysis and designatedA45. In order to minimize the possibility of mutations introduced byPCR, a BamHI fragment from A45, which spanned the joint between the CNTFand transferrin gene sequences, was cloned into BamHI digested g*. Theresulting plasmid was designated gA.

C. CNTF-(Gly)₅ -Transferrin Fusion

The CNTF-transferrin fusion separated by a series of nucleotides whichencode five glycine residues was similarly constructed using two primerpairs and templates J6 and pcDNAI/AmpNHT. Primers P33.1 (SEQ. ID. NO.20) and P53.2 (below) were complementary to the 5' and 3' ends of theCNTF gene. In addition, P53.2 contained an intervening nucleotidesequence encoding five glycines. Primers P53.1 (below) and P36.1 (SEQ.ID. NO. 23) were complementary to the 5' and 3' ends of the transferringene. P53.1 also contained the nucleotide sequence encoding fiveglycines and this sequence overlapped with the 3' ends of the fragmentgenerated with primers P33.1 and P53.2.

    P53.2                                                                              5' end of transferrin / Gly.sub.5                                           5'-CTCACAGTTTTATCAGGGAC CCCTCCACCTCC (SEQ.ID.NO.27)                           -     /   3' end of CNTF                                                      CCC CATTTTCTTGTTGTTAGC-3'                                                     - P53.1        3' end of CNTF/    Gly.sub.5       /                           5'-GCTAACAACAAGAAAATG GGGGGAGGTGGAGGG GT (SEQ.ID.NO.28)                       -  5' end of transferrin                                                      CCCTGATAAAACTGTGAG-3'                                                  

After the first PCR reaction, the two amplified fragments were purified,annealed and subjected to a second round of PCR using primers P33.1 andP36.1. The final product was digested with BlnI and EagI and ligatedwith NheI and EagI digested CD5lneg1 DNA. A clone was identified byrestriction analysis and designated B45. The BamHI fragment was againexchanged for the BamHI fragment in g*, resulting in a plasmiddesignated gB.

Competition assays for transferrin receptor binding activity wereperformed as described in Example 11 to measure the affinity of theCNTF-transferrin fusion proteins for the human transferrin receptor. Theresults of these assays demonstrated that the CNTF-transferrin fusionproteins bind well to the human transferrin receptor.

A cell-based bioassay described by Collins et al. (1989, Brain Res. 502:pp. 99-108) was used to assay CNTF activity in the CNTF-transferrinfusions by measuring the ability of these fusion proteins to stimulateoutgrowth of isolated neurons. The results of these bioassaysdemonstrated that all fusion proteins retained CNTF activity.

EXAMPLE 14 Construction of NGF-Anti-Transferrin Receptor Antibody FusionGenes, Expression of NGF-Anti-Transferrin Receptor Antibody Fusion Genesas Fusion Proteins and Assay of the Fusion Protein Constituents

Fusion proteins were constructed which were comprised of human NGF and achimeric antibody that recognizes the human transferrin receptor. TheNGF sequence was joined to the N-terminus of the heavy chain of theantibody molecule through a short linker segment consisting of twoalanine residues. As described below, separate expression plasmidsencoding the light chain and the heavy chain of the antibody and theirfusion derivatives were initially constructed to facilitate screening bytransient transfection. Subsequently, the heavy and light chainsequences were incorporated into a single expression plasmid.

Preparation of Antibody Light Chain Expression Plasmids

The vector pRC/CMVL was used for the construction of antibody lightchain mammalian expression plasmids. This vector is a derivative of thepRC/CMV plasmid (Invitrogen) which has been modified to carry a largerversion of the cytomegalovirus (CMV) major immediate early (MIE)promoter, by inserting a MluI-HindIII fragment derived from pEE14(Celltech, Ltd.) into the MluI-HindIII digested pRC/CMV plasmid.

The initial construct, pCMVκ12, contained the light chain variableregion of the murine 128.1 anti-human transferrin receptor monoclonalantibody (see Example 10) fused directly to a human kappa light chainconstant domain. One way to construct this plasmid is to ligate togethera HindIII-PpuMI fragment derived from the plasmid pAG4611 (WO 93/10819published Jun. 10, 1993; pertinent portions of which are hereinincorporated by reference) containing the sequence coding for the 128.1variable region, along with two adjacent fragments encoding the humankappa constant region, into HindIII and XbaI cleaved pRC/CMVL. The twofragments encoding the human kappa constant region are derived frompAG4611, but are modified to contain an XbaI site immediately followingthe 3' end of the kappa coding region. Introduction of the Xbal site maybe performed in the following manner.

A PCR primer pair consisting of one primer that is complementary to the5' end of the kappa sequence just upstream of an XmnI site, and a secondprimer that is complementary to the 3' end of the kappa sequence andcontains an XbaI restriction site, is used to generate a fragment of thehuman kappa constant region.

    (SEQ.ID.NO.29)                                                                  Primer A 5'CTGTTGTGTGCCTGCTGAATA-3'                                            -                                                                          (SEQ.ID.NO.30)                                                                                           XbaI     stop                                        Primer B 5'GCGTACGTACG TCTAGA AAC TAA CACTCATT                                 CCTGTTGAA-3'                                                           

The PCR generated fragment amplified off the pAG4611 template isdigested with XmnI and XbaI and ligated along with a PpuMI-XmnI fragmentfrom pAG4611 (containing the 5' end of the kappa constant region codingsequence) and a HindIII-PpuMI fragment from pAG4611 into HindIII andXbaI cleaved pRC/CMVL creating PCMVκ12. This plasmid was used togenerate pCMVκIVS21, which restored the intron present between thevariable and constant domain coding exons. This plasmid was constructedby exchanging a 0.4 kb PpuMI-XmnI fragment from pCMVκ12 with theintron-containing 3.0 kb PpuMI-XmnI fragment derived from pAG4611.

Preparation of Antibody Heavy Chain and NGF-Heavy Chain FusionExpression Plasmids

The chimeric antibody sequences used in the construction of the heavychain expression plasmids were derived from the plasmid pAH4602 (WO93/10819 published Jun. 10, 1993; pertinent portions of which are hereinincorporated by reference) and consisted of the heavy chain variableregion of the murine 128.1 anti-human transferrin receptor monoclonalantibody fused to human genomic DNA encoding the IgG1 constant region. A3.0 kb EcoRV-BamHI fragment of pAH4602 carrying the complete chimericheavy chain gene was cloned into the mammalian expression vector pEE13(Celltech, Ltd.) between the SmaI and BclI sites, creating the plasmidpEEγ1. This plasmid was then modified to contain a unique HindIII siteimmediately following the promoter by eliminating a second HindIII sitedownstream of the heavy chain coding region. The second HindIII site waseliminated by using DNA polymerase to fill in a HindIII partiallydigested plasmid followed by religation. This pEEγ1 derivative wasdesignated pEEγHIIIb.

The plasmid which expresses human NGF joined to the amino terminus ofthe antibody heavy chain was constructed using the following multi-stepprocedure. The 5' untranslated region and the segment which encodes thesignal sequence of the chimeric 128.1 heavy chain were deleted from thepEEγ1HIIIb plasmid and replaced with a NotI site-containing linkersegment. This linker segment encodes a pair of alanine residues in framewith the mature amino terminus of the heavy chain variable region. Thisreplacement was done using PCR-mediated mutagenesis to generate themodified DNA fragment which was then exchanged into the pEEγ1HIIIbplasmid between the HindIII and NheI sites. Mutagenesis was carried outusing pAH4602 as the template and oligonucleotides #375 and #376 as the5' and 3' PCR primers, respectively. Primer #375 was complementary tothe 5' end of the 128.1 heavy chain gene with the addition upstream of aHindIII site followed by the NotI (Ala-Ala) linker. Primer #376 wascomplementary to a segment found in the CH1-encoding portion of theantibody gene.

                HindIII    NotI                                                     #375 5'-GGGG AAGCTT TT GCGGCCGC TGAGGTCCAGCTGCAAC (SEQ.ID.NO.31)                                                 AGTCTG-3'                                   - #376 5'-CCGCTGGTCAGGGCGCCTGAGTT-3' (SEQ.ID.NO.32)                    

The amplified PCR fragment was digested with HindIII and NheI and thenligated with the HindIII-NheI vector fragment isolated from pEEEγ1HIIIb.Following transformation into the E.coli strain XL-1 Blue, a clonecontaining the modified 128.1 heavy chain gene was identified byrestriction digestion and verified by DNA sequencing. This plasmid,pEEγ1HIIIb/NotI, served as an intermediate in the construction of thefinal NGF-antibody fusion plasmid.

A second intermediate construct, pRC/CMVL-NFG120, containing the humanpreproNGF gene in which the 3' coding sequence was altered to introducea NotI site was generated in the following manner. In the plasmidpRC/CMVL-NGF120, the preproNGF is preceded by a Kozak sequence(described in Example 11) and is under the control of the long versionof the CMV MIE promoter (described above). One way to constructpRC/CMVL-NGF120 is to first remove the HindIII-SmaI insert frompGEM2/KNGF (Example 11) and insert it into HindIII and SmaI digestedpEE14 (Celltech, Ltd.) creating pEENGF7. An approximately 1000 base pairfragment containing the Kozak sequence-prepro NGF gene and an SV40 polyA site (present in the pEE14 vector) is removed from pEENGF7 by completedigestion with HindIII and partial digestion with BamHI. This fragmentis then inserted into HindIII and BamHI digested pRC/CMVL to createpRC/CMVL-NGF120.

In order to facilitate fusion to the antibody heavy chain,pRC/CMVL-NFG120 was then modified by introducing a NotI site at the 3'end of the coding sequence in the following manner. The PCR primer pair#219/#382 was used to generate an altered DNA fragment incorporatingthese modifications. Forward (5') primer #219 is identical to a segmentwithin the human NGF prepro-coding sequence, while reverse (3') primer#382 is complementary to the 3' end of the NGF coding sequence andcarries an additional NotI restriction site which encodes an Ala-Aladipeptide in-frame with the C-terminus of NGF.

    #219                                                                             5'GCGCCCCGGCAGCGGCGATAG-3'                                                                             (SEQ.ID.NO.33)                                       -            XbaI    NotI                                                    #382 5'-GGGGG TCTAGA GCGGCCGC TCTTCTCACAGCCTTC (SEQ.ID.NO.34)                       CTGCTG-3'                                                         

The PCR fragment amplified off the pRC/CMVL-NGF120 template was cleavedwith EcoRI and XbaI and ligated back into the EcoRI/XbaI cut plasmid.Following transformation into E.coli strain XL-1 Blue, a clonecontaining the modified NGF plasmid was identified by restrictiondigestion and verified by DNA sequencing. From this plasmid, namedpRC/CMVL-NGFNotI, a HindIII-NotI fragment carrying the entire modifiedpreproNGF gene was obtained and ligated into the HindIII and NotIdigested pEEγ1HIIIb/NotI heavy chain plasmid. The product derived fromthis ligation, plasmid pRC/CMVLγ1NGF-4, encoded the completeproproNGF-(Ala)₂ -128.1 heavy chain fusion protein.

Construction of the Combined 128.1 Light Chain/NGF-128.1 Heavy ChainExpression Plasmid

Following functional assessment in transient transfection assays usingCOS7 cells, the entire light chain transcription unit was excised fromthe pRC/CMVL vector backbone and transferred into the NGF-heavy chainexpression plasmid. To accomplish this, the light chain expressionplasmid pCMVκIVS21 was cleaved with BamHI and partially digested withBglII to isolate a 6.4 kb fragment containing the CMV promoter, thelight chain gene, and the polyadenylation/termination signals. This DNAsegment was ligated into pRC/CMVLγ1NGF-4 which had been cleaved withBamHI and treated with alkaline phosphatase. After transformation intoE.coli and screening by PCR and restriction digestion, plasmids wereidentified which contained the light chain unit inserted in either ofthe two possible orientations into the BamHI site. These two plasmidconstructs were referred to as pEEAK-30κ/γ1NGF5-4 with both the heavyand light chain gene transcription units in the same orientation andpEEAK-30γ1NGF/κ5-12 where the heavy and light chain genes areconvergently transcribed.

Assay for NGF in the NGF-Antibody Fusion Protein Expressed in MammalianCells.

Expression plasmid pEEAK-30γ1NGF/κ5-12 was transfected and transientlyexpressed in COS cells. The NGF-anti-transferrin receptor antibodyfusion protein (designated NAK) in culture supernatants was detected bystandard ELISA procedures.

Briefly, capture antibody (anti-human IgG, Vector Labs), which wasspecific for human IgG1 was coated in the wells of a 96-well plate. Thewells were washed (PBS-0.05% Tween), blocked with 1 bovine serum albumin(BSA), and supernatants from transfected COS cells were added to thewells (typically in serial four-fold dilutions) and incubated for onehour at room temperature.

An anti-NGF detection antibody (rat monoclonal antibody 1G3) was chosenwhich would recognize the NGF portion of the NAK fusion protein and wasadded to the wells on top of the fusion protein. Bound antibody wasdetected by peroxidase reaction after amplification of the signals by anavidin-biotin reaction using biotinylated anti-rat antibody (VectorLabs)and the Vectastain ABC kit (VectorLabs). Protein was quantitated byextrapolation from standard curves generated for known concentrations ofNGF.

Alternative ELISA procedures may be used to detect and quantitate thefusion proteins. For example, the capture antibody and detectionantibody may recognize the same portion of the fusion protein.

To produce stable cell lines expressing NAK, linearized vectorpEEAK-30γ1NGF/κ5-12 was electroporated into CHO cells and the cells weresubjected to drug selection in 25 mM methionine sulfoximine (MSX).Expression of the NAK fusion protein was assayed by ELISA as describedabove. Proteins containing both the NGF and anti-transferrin receptorantibody sequences were expressed and detected in the culturesupernatant.

Purification of NGF-Anti-Transferrin Receptor Antibody Fusion Proteinfrom COS Cell Supernatants.

NGF-anti-transferrin receptor antibody fusion protein present in thesupernatant of transfected COS cells was purified by using a Protein Aaffinity column (Perseptive Biosystems, Cambridge, Mass.) followed by aPoros HS/50 cation exchange column (Perseptive Biosystems) according tothe manufacturer's instructions.

The affinity purified fractions contained two major bands at 63 kD and24 kD MW on a reducing SDS-polyacrylamide gel. Both bands wererecognized by anti-human IgG1 antibody. The 63 kD band was alsorecognized by an anti-NGF antibody.

In vitro Competition Assay for Transferrin Receptor Binding Activity

A critical attribute of the NAK fusion protein is its ability to bind tothe transferrin receptor. Assays were performed to measure the affinityof the NAK fusion protein for the human transferrin receptor by theability of the fusion protein to compete with native anti-transferrinreceptor antibody (128.1) for binding to the transferrin receptor.

Competition assays were performed as in Example 11 with the exceptionsthat soluble human transferrin receptor was used and a mixture of 5 mMhorseradish peroxidase labeled anti-transferrin receptor antibody andvarying concentrations of NAK fusion protein were applied to each well.The results of these competition assays demonstrated that the NAK fusionprotein binds to the human transferrin receptor with a slightly reducedaffinity compared to the anti-transferrin receptor antibody.

In vitro Assay for NGF Activity.

A cell-based bioassay using cell line 6-24 (obtained from David Kaplan,Frederick Cancer Research Center, Frederick, Md.) was used to assesswhether the expressed NAK fusion protein has NGF activity by measuringits ability to stimulate neurite outgrowth from these cells. The 6-24cell line is derived from PC12 cells that have been engineered tooverexpress the trkA high affinity NGF receptor by transfecting the PC12cells with a trkA expression vector.

The 6-24 cells were grown in DMEM medium (Bio Whittaker) containing 5%fetal calf serum, 10% horse serum and 2 mM L-glutamine, in T75 flasksunder 5% CO₂. Ninety-six well plates were coated with 1.0 μg/cm² bovinecollagen IV in 0.05N HCl at 50 μl/well for 1 hour at room temperatureand washed 3 times with PBS. Five ml of 6-24 cells were removed fromeach flask and forced through a 21 g needle about 2-5 times to break upclumps. This procedure caused the cells to lose their neurites. Thecells were diluted with media to approximately 2×10⁴ cells/ml, 50 μlwere added to each well of the collagen-coated plates (1000 cells/well),and incubated overnight at 37° C. to allow the cells to attach.

Samples to be tested were filter sterilized before use. To generate adose-response curve, the samples were serially diluted in two-foldincrements in growth medium and 50 μl samples were added to the wells.Purified mouse NGF was serially diluted and plated in the same manner togenerate a standard curve. After 1 day exposure to the NGF-containingsamples, plates were scored for the presence or absence of NGF activityby counting the total number of cells and the number of cells sproutingat least one neurite that is longer than twice the diameter of the cellbody in two or three representative fields of view. The results wereexpressed as the percent of cells extending neurites as a function ofNGF concentration. The results of this assay demonstrated that thefusion protein fully retained NGF biological activity in vitro.

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments expressly described herein. These are intended to be withinthe scope of the invention as described by the claims herein.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 34                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  28 base - # pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - GGGTCGACCT CGAGGGTGAG AGGCCAGC         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - GGAGTTACTC AGATCTGGGA AG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - GAAGCTTCTC GAGTCTAGAC CAGGTGCATA GCGTAATGTC C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - CGTCGACCTC GAGTCTCACA GCCTTCCTGC TGAGC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - CGTCGACTCT AGATTATCTC ACAGCCTTC         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - TCCCCCGGGT CTAGACCAGG TGCATCCACC ATGTCCATGT TGTTC   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - AACAGCTATG ACCATG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1459 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                               - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: CD5lneg1                                                 - -   (viii) POSITION IN GENOME:                                                       (A) CHROMOSOME/SEGMENT: CD - #5 Leader; IgG1 Exon 1; IgG1 Exon                     2; IgG1 - #Exon 3                                                        (B) MAP POSITION: 97-17 - #7; 535-593; 698-1027; 1124-1444           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - CTCGAGATCC ATTGTGCTCT AAAGGAGATA CCCGGCCAGA CACCCTCACC TG -             #CGGTGCCC     60                                                                 - - AGCTGCCCAG GCTGAGGCAA GAGAAGGCCA GAAACCATGC CCATGGGGTC TC -            #TGCAACCG    120                                                                 - - CTGGCCACCT TGTACCTGCT GGGGATGCTG GTCGCTTCCG TGCTAGCGGA TC -            #CCGAGGGT    180                                                                 - - GAGTACTAAG CTTCAGCGCT CCTGCCTGGA CGCATCCCGG CTATGCAGCC CC -            #AGTCCAGG    240                                                                 - - GCAGCAAGGC AGGCCCCGTC TGCCTCTTCA CCCGGAGCCT CTGCCCGCCC CA -            #CTCATGCT    300                                                                 - - CAGGGAGAGG GTCTTCTGGC TTTTTCCCAG GCTCTGGGCA GGCACAGGCT AG -            #GTGCCCCT    360                                                                 - - AACCCAGGCC CTGCACACAA AGGGGCAGGT GCTGGGCTCA GACCTGCCAA GA -            #GCCATATC    420                                                                 - - CGGGAGGACC CTGCCCCTGA CCTAAGCCCA CCCCAAAGGC CAAACTCTCC AC -            #TCCCTCAG    480                                                                 - - CTCGGACACC TTCTCTCCTC CCAGATTCCA GTAACTCCCA ATCTTCTCTC TG -            #CAGAGCCC    540                                                                 - - AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG GTAAGCCAGC CC -            #AGGCCTCG    600                                                                 - - CCCTCCAGCT CAAGGCGGGA CAGGTGCCCT AGAGTAGCCT GCATCCAGGG AC -            #AGGCCCCA    660                                                                 - - GCCGGGTGCT GACACGTCCA CCTCCATCTC TTCCTCAGCA CCTGAACTCC TG -            #GGGGGACC    720                                                                 - - GTCAGTCTTC CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GG -            #ACCCCTGA    780                                                                 - - GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT TC -            #AACTGGTA    840                                                                 - - CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG CGGGAGGAGC AG -            #TACAACAG    900                                                                 - - CACGTACCGG GTGGTCAGCG TCCTCACCGT CCTGCACCAG GACTGGCTGA AT -            #GGCAAGGA    960                                                                 - - GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CC -            #ATCTCCAA   1020                                                                 - - AGCCAAAGGT GGGACCCGTG GGGTGCGAGG GCCACATGGA CAGAGGCCGG CT -            #CGGCCCAC   1080                                                                 - - CCTCTGCCCT GAGAGTGACC GCTGTACCAA CCTCTGTCCT ACAGGGCAGC CC -            #CGAGAACC   1140                                                                 - - ACAGGTGTAC ACCCTGCCCC CATCCCGGGA TGAGCTGACC AAGAACCAGG TC -            #AGCCTGAC   1200                                                                 - - CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GC -            #AATGGGCA   1260                                                                 - - GCCGGAGAAC AACTACAAGA CCACGCCTCC CGTGCTGGAC TCCGACGGCT CC -            #TTCTTCCT   1320                                                                 - - CTACAGCAAG CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TC -            #TCATGCTC   1380                                                                 - - CGTGATGCAT GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TG -            #TCTCCGGG   1440                                                                 - - TAAATGAGTG CGACGGCCG             - #                  - #                     145 - #9                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - AAGGAGGTGA TGGTGTTGGG A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - CTCACAGTTT TATCAGGGAC TCTCACAGCC TTCCTGCTGA GC    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - CAGCAGGAAG GCTGTGAGAG TCCCTGATAA AACTGTGAGA TG    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - GTGTGGCAGG ACTTCTTGCC T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - CAGCAGGAAG GCTGTGAGAG GGGGAGGTGG AGGGGTCCCT GATAAAACTG TG - #AGATG            57                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - CTCACAGTTT TATCAGGGAC CCCTCCACCT CCCCCTCTCA CAGCCTTC  - #                    48                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - GGAACGGCCT CGAGGTCCCT GATAAAACTG TGAGA       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - AGTTAACATA TGGCTTTTAC TGAGCATTCA C        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - CAGGCCCTGA TGCTTCACAT AGGATTCCGT AAGAGCAGT      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - TACGGAATCC TATGTGAAGC ATCAGGGCCT GAACA       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - GGGCCCTCGA GGGACTAACT GCTACATTTT CTTGTTGTTA GC    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - CGCGGGCCTA GGCGCTTTCA CAGAGCATTC ACC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - CGCGGGGCGG CCGCTTTACA TTTTCTTGTT GTTGTTAG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - GCTTCCGTCC TAGGGGTCCC TGATAAAACT GTG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - CGCGGGGCGG CCGCTTTAAG GTCTACGGAA AGTGCA      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - TGGCCTCTCA CCCTCGAGCA TTTTCTTGTT GTTAGC      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - CTCACAGTTT TATCAGGGAC CATTTTCTTG TTGTTAGC      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - GCTAACAACA AGAAAATGGT CCCTGATAAA ACTGTG      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                          - - CTCACAGTTT TATCAGGGAC CCCTCCACCT CCCCCCATTT TCTTGTTGTT AG - #C                53                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                          - - GCTAACAACA AGAAAATGGG GGGAGGTGGA GGGGTCCCTG ATAAAACTGT GA - #G                53                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                          - - CTGTTGTGTG CCTGCTGAAT A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                          - - GCGTACGTAC GTCTAGAAAC TAACACTCAT TCCTGTTGAA     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #31:                          - - GGGGAAGCTT TTGCGGCCGC TGAGGTCCAG CTGCAACAGT CTG    - #                      - # 43                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                          - - CCGCTGGTCA GGGCGCCTGA GTT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 33:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                          - - GCGCCCCGGC AGCGGCGATA G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 34:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #34:                          - - GGGGGTCTAG AGCGGCCGCT CTTCTCACAG CCTTCCTGCT G    - #                      - #   41                                                                    __________________________________________________________________________

We claim:
 1. A fusion protein comprising a brain capillary endothelialcell receptor ligand and a neuropharmaceutical agent wherein:(a) theneuropharmaceutical agent consists of nerve growth factor (NGF), and (b)the ligand is selected from the group consisting of transferrin,insulin, an antibody to the transferrin receptor, an antibody toinsulin-like growth factor 1 (IGF 1) receptor, an antibody toinsulin-like growth factor 2 (IGF 2) receptor and an antibody to insulinreceptor; wherein the ligand and the neuropharmaceutical agent arelinked through an intermediate peptide or polypeptide.
 2. A fusionprotein comprising a brain capillary endothelial cell receptor ligandand a neuropharmaceutical agent wherein:(a) the neuropharmaceuticalagent consists of nerve growth factor (NGF), and (b) the ligand is anantibody to the transferrin receptor; wherein the ligand and theneuropharmaceutical agent are linked through an intermediate peptide orpolypeptide.
 3. A fusion protein comprising a brain capillaryendothelial cell receptor ligand and a neuropharmaceutical agentwherein:(a) the neuropharmaceutical agent consists of nerve growthfactor (NGF), and (b) the ligand is transferrin;wherein the ligand andthe neuropharmaceutical agent are linked through an intermediate peptideor polypeptide.
 4. A fusion protein comprising a brain capillaryendothelial cell receptor ligand and a neuropharmaceutical agentwherein:(a) the neuropharmaceutical agent consists of ciliaryneurotrophic factor (CNTF), and (b) the ligand is transferrin;whereinthe ligand and the neuropharmaceutical agent are linked through anintermediate peptide or polypeptide.
 5. A fusion protein comprising abrain capillary endothelial cell receptor ligand and aneuropharmaceutical agent wherein:(a) the neuropharmaceutical agent isselected from the group consisting of a growth factor, superoxidedismutase, CD4, a lymphokine, a lymphokine antagonist, a cytokineantagonist, dopamine decarboxylase and tricosanthin; and (b) the ligandis selected from the group consisting of transferrin, insulin, anantibody to the transferrin receptor, an antibody to insulin-like growthfactor 1 (IGF 1) receptor, an antibody to insulin-like growth factor 2(IGF 2) receptor and an antibody to insulin receptor;wherein the ligandand the neuropharmaceutical agent are linked through an intermediatepeptide consisting of leucine-glutamic acid (leu-glu).
 6. The fusionprotein of claim 5 wherein the ligand consists of transferrin and theneuropharmaceutical agent is nerve growth factor (NGF).